Method of identifying transmembrane protein-interacting compounds

ABSTRACT

A method for screening compounds for their ability to interact with transmembrane proteins is provided. Also provided is a method for determining whether proteins such as transmembrane proteins are able to oligomerize.

RELATED APPLICATION INFORMATION

The present application is a 35 U.S.C. § 371 National Phase Applicationof International Application Serial No. PCT/CA03/00542, filed on Apr.11, 2003 and published in English as PCT Publication No. WO 03/087836,which claims priority from U.S. Provisional Application Ser. No.60/371,704, filed Apr. 12, 2002, U.S. Provisional Application Ser. No.60/379,419, filed May 13, 2002, U.S. Provisional Application Ser. No.60/387,570, filed Jun. 12, 2002, Ser. No. 60/422,891, filed Nov. 1,2002, and U.S. Provisional Application Ser. No. 60/442,556, filed Jan.27, 2003, the disclosures of each of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to methods for screening compounds for theirability to interact with transmembrane proteins. The invention furtherrelates to methods for screening transmembrane proteins for theirability to dimerise or oligomerise into groups of two or more proteins.

BACKGROUND OF THE INVENTION

In the description which follows, references are made to certainliterature citations which are listed at the end of the specificationand all of which are incorporated herein by reference.

Transmembrane proteins have been classified in several major classes,including G protein coupled receptors, transporters, tyrosine kinasereceptors, cytokine receptors and LDL receptors. G protein coupledreceptors (GPCRs) can be grouped on the basis of structure and sequencehomology into several families. Family 1 (also referred to as family Aor the rhodopsin-like family) is by far the largest subgroup andcontains receptors for small molecules such as the catecholamines,dopamine and noradrenaline, peptides such as the opioids, somatostatinand vasopressin, glycoprotein hormones such as thyrotropin stimulatinghormone and the entire class of odorant molecules (George et al, 2002).Family 2 or family B contains the receptors such as for glucagon,parathyroid hormone and secretin. These GPCRs are characterised by along amino terminus that contains several cysteines, which may formdisulphide bridges. Family 3 or family C contains receptors such as themetabotropic glutamate, the Ca2+-sensing and the gamma-amino butyricacid (GABA)B receptors. These receptors are also characterised by acomplex amino terminus. Although all GPCRs share the sevenmembrane-spanning helices, the various GPCR families show no sequencehomology to one another.

GPCRs are the largest known group of cell-surface mediators of signaltransduction and are present in every cell in the body. GPCR actionregulates the entire spectrum of physiological functions, such as thoseinvolving the brain, heart, kidney, lung, immune and endocrine systems.Extensive efforts during the past decade has identified a large numberof novel GPCRs, including multiple receptor subtypes for previouslyknown ligands, and numerous receptors for which the endogenous ligandsare as yet unidentified, termed ‘orphan’ receptors or oGPCRs (Lee etal., 2001; Lee et al., 2002; Bailey et al., 2001).

GPCRs have been the successful targets of numerous drugs for diversedisorders in clinical use today, with an estimated 50% of the currentdrug market targeting these molecules. Among the known GPCRs, ˜335receptors are potential drug development targets, of which 195 haveknown ligands, and the remaining 140 being oGPCRs, awaitingidentification of their ligands. Although various methodologicaladvances have accelerated the pace of novel receptor discovery, the paceof ligand and drug discovery lags far behind. Conventional, small-scalepharmacological screening assay methods were initially used to discoverthe ligands and drugs for many of the GPCRs, but newer assay proceduresare continually being sought.

Since GPCRs form over 80% of cell surface receptors, they represent asubstantial resource and constitute a highly relevant group of proteintargets for novel drug discovery. Drugs interacting with GPCRs have thepotential to be highly selective, as the interactions will be confinedto the cell surface and to tissues bearing the receptors exclusively.The convergence of the discovery of GPCRs with the realisation that theyare important drug targets, has led to intense pharmaceutical interestin devising better ways to detect and screen for compounds interactingwith GPCRs. Therefore, creating improved assay methods is an urgentrequirement towards the goal of more rapid drug screening and discovery.There is a need to optimise the ability to detect an interaction betweentest compounds and the receptors, which is the fundamental initial stepin the process of drug development.

Improved ligand-identification strategies to accelerate thecharacterisation of all GPCRs will define their physiological functionsand realise their potential in discovering novel drugs. Even with theidentified GPCRs, there is a paucity of highly selective subtypespecific drugs being discovered and pharmaceutical houses areexperiencing a dearth of promising lead compounds, in spite of thewealth of drug targets defined. The list of new drug product approvalsby the top 20 pharmaceutical companies has declined considerably overthe period 1999-2001, compared to the preceding three year period(Smith, 2002). Thus there is a real need to have improved, versatileassay systems, where not just endogenous ligands, but novel compoundsinteracting with receptors can be tested and identified in a quick andefficient manner that is amenable to automation.

As the signal transduction pathway required to activate an oGPCR cannotbe predicted, an assay system for interacting compounds which isindependent of prior predictions of which effector system (such asadenylyl cyclase, PLC, cGMP, phosphodiesterase activity) is employed bythe receptor is required. Assigning ligands to GPCRs and oGPCRs is animportant task; however the diversity of both GPCR ligands and effectorsystems can limit the utility of some existing ligand-identificationassays, requiring novel approaches to drug discovery.

Recently, several methods utilising refined assay systems testing tissueextracts, large ligand libraries and specific ligands of interest havesuccessfully discovered the endogenous ligands for a number of theseoGPCRs. Such methods have been collectively referred to as “ReversePharmacology” (Howard et al., 2001). Various methods have been used toassay induced cell activity in response to an agonist compound,including the Fluorescence Imaging Plate Reader assay (FLIPR, MolecularDevices Corp., Sunnyvale, Calif.) and Barak et al., (1997), and U.S.Pat. Nos. 5,891,646 and 6,110,693 which disclose the use of aβ-arrestin-green fluorescent fusion protein for imaging arrestintranslocation to the cell surface upon stimulation of a GPCR.

The potential disadvantages of such methods are as follows: 1)visualisation is not of the receptor; 2) the protein translocationrequires complex computerised analytical technologies; 3) prioridentification of agonist is necessary to screen for antagonists; and 4)specific G protein coupling is necessary to generate a signal.

Mechanisms of ligand binding and signal transduction by GPCRstraditionally have been modelled on the assumption that monomericreceptors participate in the process, and a monomeric model for GPCRshas been generally accepted. Since the mid-1990s, however, numerousreports have demonstrated oligomerisation of many GPCRs (reviewed byGeorge et al., 2002), and it is now realised that oligomerisation is aninherent aspect of GPCR structure and biology. Also certain receptorsubtypes formed hetero-oligomers, and these receptors have functionalcharacteristics that differ from homogeneous receptor populations. Atpresent, studies of GPCR oligomerisation do not make a distinctionbetween dimers and larger complexes, and the term dimer is usedinterchangeably with the terms oligomer and multimer. There are noconclusive data to indicate how large the oligomers of functional GPCRsare. Importantly, generation of new properties throughhetero-oligomerisation suggested a mechanism for generating diversity offunction among GPCRs. Homooligomerisation of GPCRs is accepted as auniversal occurrence and a number of GPCRs are known to assemble asheterooligomeric receptor complexes (George et al., 2002). For example,the GABA-B1 and GABA-B2 receptors are not functional individually andonly form a functional receptor when co-expressed (White et al., 1998).The assembly of heterooligomer receptor complexes can result in novelreceptor-ligand binding, signalling or intracellular traffickingproperties. For example, co-transfection of the mu and delta opioidreceptors resulted in the formation of oligomers with functionalproperties that were distinct from each of the receptors individually(George et al., (2000). The interaction of mu and delta opioid receptorsto form oligomers generated novel pharmacological and G protein couplingproperties. When mu and delta opioid receptors were co-expressed, thehighly selective agonists (DAMGO, DPDPE, and morphine) had reducedpotency and altered rank order, whereas certain endogenous ligandsendomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting theformation of a novel ligand binding pocket (George et al., 2000). Incontrast to the individually expressed mu and delta receptors, thecoexpressed receptors showed pertussis toxin insensitive signaltransduction, likely due to interaction with a different subtype of Gprotein. It would therefore be very useful, from the point of view ofidentification of potential drug targets, to have a means of determiningwhether a particular pair of GPCRs are able to form heterooligomers.

In many reports, heterooligomers have been tentatively identified by theability to co-immunoprecipitate. When two GPCRs are shown toco-immunoprecipitate, however, there are two possible interpretations;either the receptors are directly physically interacting, or both areinteracting through contact with a common third protein (or proteins).An alternative approach to detecting receptor oligomers has been thedevelopment of energy transfer assays using bioluminescent resonanceenergy transfer (BRET) or fluorescence resonance energy transfer (FRET).Although these methods detect energy transfer between two receptormolecules labelled by fluorophores at proximities of less than 100angstroms, it is unclear whether receptor conformational changes can bereliably distinguished from de novo oligomerisation.

Transporters are protein pumps that move molecules, ions and otherchemicals in and out of cells and exist in virtually all cells. Thetransporters can be grouped into families on the basis of structure,sequence homology and the molecules they transport. Separatetransporters exist for monoamine neurotransmitters such as dopamine,serotonin, norepinephrine and GABA, for amino acids such as glycine,taurine, proline and glutamate, for vesicular monoamines, acetylcholineand GABA/glycine, for sugars such as glucose and disaccharides, fororganic cations and organic anions, for oligopeptides and peptides, forfatty acids, bile acids, nucleosides, for water and for creatine. Pumpsthat export large molecules such as drugs, toxins and antibiotics fromthe cell are exemplified by the P-glycoprotein (multidrug resistanceprotein) family. There are also several related transporters thefunction of which remains unknown (Masson et al., 1999). Thesetransporters are membrane proteins consisting of a polypeptide generallywith 12 transmembrane domains. The glutamate and aspartate transportersbelong to a separate family whose members have 6 to 10 TM domains andshare no homology to the other transporters (Masson et al., 1999). Boththe amino and carboxyl termini are located on the intracellular side ofthe membrane.

A large number of neurological and psychiatric disorders includingdepression, Parkinson's disease, schizophrenia, drug addiction,Tourette's syndrome, and attention deficit disorders are considered toinvolve the monoamine transporters. The dopamine transporter (DAT) isthe major target for psychostimulants such as cocaine andmethylphenidate. The transporters have been the successful targets ofnumerous drugs for diverse disorders in clinical use today, particularlyantidepressant drugs, including fluoxetine, sertraline and the otherrelated serotonin selective reuptake inhibitors (SSRIs). Althoughmethodological molecular advances have identified the knowntransporters, the pace of ligand and drug discovery lags behind.Conventional, pharmacological screening assay methods were used todiscover the ligands and drugs for some of the transporters, but newerassay procedures are urgently being sought. Improvedligand-identification strategies to accelerate the characterisation ofall the transporters will further define their physiological functionsand realise their potential in discovering novel drugs. Even with theidentified transporters, there is a paucity of highly selective specificdrugs being discovered.

The tyrosine kinase receptor family members are characterised by theirstructural similarity, with an extracellular ligand binding domain, asingle transmembrane domain and an intracellular domain with tyrosinekinase activity for signal transduction. There are many subfamilies ofreceptor tyrosine kinases, exemplified by the epidermal growth factor(EGF) receptor (also called HER1 or erbB1), which is one of four membersof such a subfamily, which also includes HER2, HER3 and HER4. Theprincipal EGF-R ligands are EGF, TGF-α, heparin binding EGF,amphiregulin, betacellulin and epiregulin (Shawver et al., 2002).Activation of the EGF-R causes the receptor to dimerise with eitheranother EGF-R monomer or another member of the HER subfamily. Markeddiversity of ligand binding and signalling is generated by the formationof heterodimers among family members (Yarden and Sliwkowski, 2001). TheEGF-R is widely expressed in a variety of tissues and mediates importantfunctions such as cell growth and tissue repair. Overexpression of EGF-Roccurs in many types of cancer, such as head and neck, lung, laryngeal,esophageal, gastric, pancreatic, colon, renal, bladder, breast, ovarian,cervical, prostate, thyroid, melanoma and glioma, and correlates with apoor outcome (Nicholson et al., 2001). Therefore there is great interestand need for developing drugs targeting the EGF-R and for methods whichassist in identifying such drugs.

Other subfamilies of receptor tyrosine kinases are exemplified by thereceptors for vascular endothelial factor (four members) and fibroblastgrowth factor (four members). These have important roles in angiogenesisand also have significant roles in the uncontrolled proliferation ofvessels characterizing carcinogenesis (Hanahan and Folkman. 1996).

The cytokine receptors are proteins spanning the membrane with anextracellular ligand binding domain and an intracellular domain withintrinsic kinase activity or adapter regions able to interact withintracellular kinases. The receptors are divided into subclasses basedon their structural complexity. The ‘simple’ receptors are thoseincluding receptors for growth hormone, erythropoietin and interleukins,and the ‘complex’ receptors include the tumour necrosis factor receptorfamily, the 4-helical cytokine receptor family, the insulin/insulin-likereceptor family and granulocyte colony stimulating receptor (Grotzinger,2002).

The insulin and insulin-like growth factor (IGF) receptor familycontrols metabolism, reproduction and growth (Nakae et al., 2001). Thereare nine different insulin-like peptides known and there are three knownreceptors that interact with them, IR, IGF-1R and IGF-2R, and an orphanmember IR-related receptor. Each receptor exists as homodimers on thecell surface or heterodimers. The IR subfamily is also related to theEGF-R family.

IR, produced from a single mRNA, undergoes cleavage and dimerisation andtranslocation to the plasma membrane. Each monomer component contains asingle transmembrane domain; the complete receptor comprised two α andtwo β subunits, linked by disulphide bridges. The β subunit contains thesingle TM and the intracellular region. This receptor is a tyrosinekinase that catalyzes the phosphorylation of several intracellularsubstrates.

The low density lipoprotein (LDL)-receptor family act as cargotranporters, regulating the levels of lipoproteins and proteases(Strickland et al., 2002). There are nine recognised members of thefamily, all of which share structural similarity, including anextracellular region, a single transmembrane domain region and acytoplasmic tail. The LDL receptor plays a major role in the clearanceof lipoproteins, and genetic defects in the LDL receptor can result inthe accumulation of LDL in the bloodstream.

The first characterised motif shown to be able to direct protein nuclearimportation was exemplified by the amino acid sequence (PKKKRKV:SEQ IDNO: 129) contained in the SV40 large T antigen protein. The nuclearlocalisation sequence (NLS) motifs are recognised by the importin α-βreceptor complex, which binds the NLS (Gorlich et al., 1996). These arecytosolic proteins, which recognise NLS containing proteins andtransport these proteins to dock at the nuclear pore. The entire complexsubsequently docks at the nuclear pore complex (Weis et al., 1998,Schlenstedt et al., 1996), contained at the nuclear envelope. Thenuclear envelope is a boundary containing pores that mediate the nucleartransport process (Weis et al., 1998).

There have been very few and rare reports of GPCRs localising in thenucleus. One such example is the GPCR angiotensin type 1 (AT₁) receptor,which contains an endogenous NLS which serves to direct the GPCR intothe nucleus (Lu et al., 1998), providing evidence that this NLS sequencewas involved in the nuclear targeting of the AT1 receptor. These authorsand Chen et al., (2000) reported that AT1 receptors increased in thenucleus in response to agonist. The nuclear localisation of theparathyroid hormone receptor has been reported (Watson et al, 2000).However very few of the superfamily of GPCRs contain an endogenous NLSmediating translocation of the receptor to the nucleus.

There therefore remains a need for new, more convenient methods foridentifying compounds which interact with transmembrane proteins such asGPCRs, transporters, etc. There also remains a need for improved, lessambiguous methods for detecting oligomerisation of transmembraneproteins.

SUMMARY OF THE INVENTION

The inventors have shown that the incorporation of a nuclearlocalisation sequence (NLS) into a transmembrane protein (not containingan endogenous functional NLS) routes the protein from the cell surfaceinto the nucleus of a cell in a time-dependent and ligand-independentmanner. In order to visualise this trafficking of transmembrane proteinsfrom the cell surface, they carry a detectable moiety for visualisationby a variety of means. It has been demonstrated that membrane proteinsfrom diverse protein families containing a synthetically incorporatedNLS are redistributed under basal conditions from the cell surface toand towards the nucleus.

This process can be exploited to identify compounds which interact withtransmembrane proteins by determining whether candidate compounds areable to modulate this ligand-independent transfer of a transmembraneprotein away from the cell membrane.

It is also now possible, using methods based on this process, todetermine whether protein molecules are able to oligomerise.

In accordance with one embodiment, the invention provides a method forscreening a candidate compound for its ability to interact with at leastone transmembrane protein comprising:

transfecting a cell with at least one nucleotide sequence encoding aprotein comprising a transmembrane protein containing at least onenuclear localisation sequence (NLS) and a detectable moiety andpermitting expression of the encoded protein in the cell;

contacting the cell with a candidate compound; and

determining the distribution of the expressed protein in the cell bydetecting the distribution of the detectable moiety in the cell;

wherein detection of an altered distribution of the detectable moiety inthe cell relative to the distribution of the detectable moiety in acontrol cell not contacted with the candidate compound indicates thatthe compound interacts with the transmembrane protein.

In accordance with a further embodiment of this method, the cell iscontacted with a compound known to interact with the at least onetransmembrane protein prior to contacting the cell with the candidatecompound and

wherein detection of an altered distribution of the detectable moiety inthe cell relative to the distribution of the detectable moiety in acontrol cell contacted with the compound known to interact with thetransmembrane protein but not contacted with the candidate compoundindicates that the candidate compound interacts with the transmembraneprotein.

In accordance with a further embodiment, the invention provides a methodfor screening a candidate compound for its ability to interact with atleast one transmembrane protein comprising:

transfecting a cell with at least one nucleotide sequence encoding anNLS-containing transmembrane protein and permitting expression of theencoded protein in the cell;

contacting the cell with a candidate compound; and

determining the level of NLS-containing transmembrane protein remainingat the cell membrane by isolating the cell membrane fraction of thecell, contacting the fraction with a labelled ligand of thetransmembrane protein and determining the level of binding of the ligandto the fraction;

wherein detection of an altered level of the transmembrane protein atthe cell membrane relative to the level at the cell membrane in acontrol cell not contacted with the candidate compound indicates thatthe compound interacts with the transmembrane protein.

In accordance with a further embodiment, the invention provides anisolated cell transfected with at least one nucleotide sequence encodinga protein comprising a transmembrane protein containing at least one NLSand a detectable moiety.

In accordance with a further embodiment, the invention provides a methodfor determining whether a first protein and a second protein are able tooligomerise comprising:

transfecting a cell with a first nucleotide sequence encoding a firstprotein containing an NLS and a second nucleotide sequence encoding asecond protein comprising a detectable moiety and permitting expressionof the encoded first and second proteins in the cell; and

determining the distribution of the detectable moiety in the cell;

wherein detection of the detectable moiety in or adjacent to the nucleusof the cell or detection of a reduced level of the detectable moiety atthe cell surface, relative to a control cell, indicates that the firstand second proteins interact.

SUMMARY OF THE DRAWINGS

Certain embodiments of the invention are described, reference being madeto the accompanying drawings, wherein:

FIG. 1 shows in diagrammatic form the structure of a typical GPCR, thedopamine D1 receptor [SEQ ID NO: 159], modified to contain an NLS.

FIG. 2 shows fluorescence (Relative fluorescence units) at surface ofHEK cells transfected with dopamine D1 receptor-NLS and treated withvarious concentrations of butaclamol.

FIG. 3 shows cell surface fluorescence of HEK cells transfected withHA-dopamine D1 receptor-NLS and treated with various concentrations ofSCH23390 alone or with SKF81297 (100 nM).

FIG. 4 shows cell surface fluorescence of HEK cells transfected withHA-dopamine D1 receptor-NLS and treated with 0.5 μM SCH23390 alone ortogether with various concentrations of SKF81297.

FIG. 5 shows the amount of ³H-SCH 23390 bound to the cell membranefraction of HEK cells transfected with dopamine D1 receptor-NLS andtreated with butaclamol (▴) or control untreated cells (▪).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in one embodiment, a new and convenient methodfor screening candidate compounds for their ability to interact with atransmembrane protein.

As used herein, when a candidate compound and a transmembrane protein“interact”, this means that the compound is a ligand of thetransmembrane protein and binds to the protein or is able to modulatethe trafficking of the transmembrane protein away from the cell membranedescribed herein.

Identification of compounds which interact with a transmembrane proteinis the first important step in the process of identifying lead compoundsfor drug development.

Working initially with GPCRs, the inventors have found that when anucleated eukaryotic cell is transfected with a nucleotide sequencewhich encodes a GPCR containing a synthetically incorporated NLS, or anaturally occurring NLS, and the cell is permitted to express thenucleotide sequence, the expressed GPCR travels first to the cellmembrane and then is transferred to the cell nucleus. This process isindependent of ligand activation and takes from about 6 to about 72hours, depending on the transmembrane protein involved, with an averageof 24 to 48 hours. This is in contrast to the situation when a GPCR notcontaining an NLS is expressed in a cell, when the expressed GPCRremains predominantly at the cell surface, with small amounts occurringin the cytoplasm but no detectable amounts in the nucleus.

The inventors have also found that the transfer or trafficking of theexpressed NLS-containing GPCR from the cell membrane to or towards thenucleus can be modulated by treating the transfected cell with acompound which interacts with the GPCR. Screening of candidate compoundsfor their ability to interact with a GPCR can therefore be carried outby detecting this modulation of transfer of the expressed GPCR from thecell membrane to the nucleus.

The inventors have further found that these observations are widelyapplicable to transmembrane proteins generally, and are not limited toGPCRs.

“Transmembrane protein” as used herein means a single chain proteinfound in the cell membrane and having at least one domain that spans thecell membrane.

The inventors have shown that a wide variety of transmembrane proteinsfrom several families of the GPCR group, from the transporter group,from the cytokine receptor group, from the tyrosine kinase group andfrom the low density lipoprotein receptor group, if expressed in anucleated cell so that they contain an NLS group, all show initialaccumulation of the expressed protein at the cell membrane, followed byligand activation-independent transfer of the expressed protein awayfrom the cell membrane and into the cell nucleus.

The wide applicability of the methods of the invention is indicated bythe immense variety of transmembrane protein structures represented bythe exemplified transmembrane proteins used in the method; NLS insertioninto a transmembrane protein resulting in translocation of the proteinoff the cell surface and to the nucleus has been shown to be effectivewith membrane proteins having one transmembrane (TM) domain, seven TMdomains and twelve TM domains and sharing little or no sequencehomology.

It has been found that the method of the invention is widely applicableto identifying compounds which interact with transmembrane proteins.

Compounds which interact with transmembrane proteins have been found tomodulate their transfer from cell membrane to nucleus in different ways,including inhibition of the transfer, acceleration of the transfer andinterference with the modulation produced by other compounds. Anyinteracting compound is of interest as a potential drug candidate.

Modulation of the transfer of expressed transmembrane protein isdetermined by comparing the distribution of the transmembrane proteinwithin the cell in control cells and cells treated with a candidatecompound.

In one embodiment, the method provides a convenient tool for screeningcandidate compounds for their ability to interact with a GPCR andmodulate its trafficking. Compounds that specifically interact with theGPCR may inhibit or prevent transfer of the GPCR from the cell surfaceto the nucleus and may be antagonists to the GPCR, whereas othercompounds can accelerate the transfer of GPCR to the nucleus, relativeto a control cell and may be agonists to the GPCR.

To allow determination of the distribution of the expressedtransmembrane protein within the cell, with and without exposure to atest compound, the expressed transmembrane protein must carry adetectable moiety, which can be detected in the cell. The detectablemoiety may be any moiety which will remain associated with transmembraneprotein throughout its expression and trafficking within the cell andcan be directly or indirectly detected to determine its distributionwithin the cell and to determine an altered distribution resulting fromexposure to a candidate compound.

In a first embodiment, the cell is transfected with a nucleotidesequence encoding a fusion protein comprising a transmembrane proteincontaining at least one NLS linked to a detectable moiety comprising adetectable peptide or polypeptide. As used herein, a peptide means asequence of two to 20 amino acid residues, preferably a sequence ofabout 5 to about 15 amino acid residues, and a polypeptide means asequence of more than 20 amino acid residues, including full proteins ofany length. The detectable peptide or polypeptide may be directlydetectable or may be reactable to give a detectable signal. Thedetectable peptide may be, for example, an antigenic peptide or epitopewhich is expressed, for example, at the amino terminus of thetransmembrane protein. The distribution of the transmembrane proteinwithin the cell is detected by detection of the epitope using adetectable antibody specific for the epitope. A number of suitableepitope antibody systems are available commercially. Examples are the HA(Roche Diagnostics), FLAG (Sigma Chemical Co.), c-myc (Santa Cruz),Histidine hexamer (BD Biosciences Clontech), GST (ABR AffinityBioReagents), V5 (Abcam) and Xpress (Invitrogen) epitope/antibodysystems.

Nucleotide sequences encoding these epitopes can be purchased, as wellas antibodies specific for the epitopes. These antibodies may carry adetectable label (e.g. fluorescein isothiocyanate (FITC)) or maythemselves be detected by use of a second antibody carrying a detectablelabel, as will be understood by those of skill in the art. Thisembodiment of the invention is particularly adaptable to an automated orsemi-automated method, for example by examining antibody-treated platesof cells in an automated plate reader, allowing for high through putscreening.

The detectable polypeptide may be an optically detectable polypeptidesuch as green fluorescent protein (GFP), red fluorescent protein (RFP),yellow fluorescent protein (YFP) and or cyan fluorescent protein (CFP),or any of the modified variants of these proteins, which arecommercially available. The detectable polypeptide may also be an enzymesuch as luciferase or β-galactosidase, which can be reacted to give adetectable end point such as light emission or colour change. Nucleotidesequences encoding such polypeptides are readily available, for examplefrom Clontech, and are linked to the nucleotide sequence encoding theNLS-containing transmembrane protein, preferably at the C-terminal endof that protein.

In a further embodiment, the detectable moiety is an antigenic peptidecomprising a portion of the amino acid sequence of the transmembraneprotein itself, preferably a portion of an extracellular region of theprotein. As described above, the distribution of the transmembraneprotein within the cell is determined using a detectable antibodyspecific for the epitope. Suitable antibodies are availablecommercially, e.g. anti-D1 antibody directed to amino terminal aminoacids 9-21 of the human D1 dopamine receptor, or may be prepared byconventional methods.

In a further embodiment, applicable to transmembrane proteins with knownligands, the cell is transfected with a nucleotide sequence encoding atransmembrane protein containing at least one NLS. The cells arecontacted with a candidate compound and incubated as described above.The cells are then harvested and the cell membrane fraction is isolatedand contacted with a detectably labelled ligand of the transmembraneprotein, for example a radio-labelled ligand. Determination of theamount of labelled ligand bound to the membrane fraction of treatedcells, relative to the amount bound to the membrane fraction of controlcells not contacted with the candidate compound, can be used toquantitate the transmembrane protein remaining at the cell surface andindicate interaction of the candidate compound with the transmembraneprotein.

Transmembrane protein-encoding nucleotide sequences can be obtained frompublic databases such as Genbank or from commercial databases. Suitableconstructs may be synthesised by conventional methods, as described inthe examples herein, or obtained commercially.

“An NLS-containing transmembrane protein” includes a transmembraneprotein which contains an NLS in its wild type sequence and atransmembrane protein whose amino acid sequence has been modified tocontain an NLS.

Conventional NLSs are short peptide sequences that facilitate nuclearlocalisation of the proteins containing them (see for example, Table 1which lists NLSs and Jans et al., 2000). There are three major classesof NLSs; two of these classes consist of basic amino acid residues, themonopartite NLSs, exemplified by the SV40 large tumor antigen, PKKKRKV(SEQ ID NO: 129), consisting of a single stretch of basic amino acids,and the bipartite NLSs which contain two stretches of basic amino acidsseparated by 10 to 22 (sometimes up to hundreds) amino acids. Othertypes of NLSs are exemplified by those of the yeast protein Mata2 NLSwhere charged/polar residues are contained within the stretch ofnon-polar residues, or the protooncogene c-myc NLS, where proline andaspartic acid residues flanking the basic residues are required(PAAKRVKLD: SEQ ID NO: 135) for nuclear targeting. All classes of NLSare recognized specifically by the α-β-importins.

Any NLS may be employed in the methods of the invention. Nucleotidesequences encoding a selected NLS may be derived from the amino acidsequence of the NLS and are synthesised and incorporated into thenucleotide sequence encoding the transmembrane protein by conventionalmethods, as described herein. Many different locations within any of theintracellular loops or intracellular termini of the transmembraneprotein are suitable for insertion of the NLS. Insertion of the NLSwithin an intracellular domain of the protein is preferred. For example,in a GPCR, the NLS could be placed in any of the intracellular loops orintracellular carboxyl tail. In a 12 TM transporter, the NLS could beplaced in the intracellular amino or carboxyl termini or any of theintracellular loops.

When an NLS is inserted into a transmembrane protein for use in themethods of the invention, the efficacy of the insertion can be screenedby confirming that the NLS-containing transmembrane protein issubstantially translocated from the cell membrane to the cell nucleuswithin 24 to 48 hours and that ligands of the transmembrane proteininterfere with the translocation.

Nucleotide sequences encoding NLS-containing transmembrane proteins arelinked to sequences encoding detectable peptides or polypeptides byconventional methods.

A nucleotide sequence encoding a selected NLS-containing transmembraneprotein containing or linked to a detectable moiety, is transfected intoa nucleated cell by cloning the sequence into a vector system containinga suitable promoter, using conventional techniques as described in thescientific literature, for example in Current Protocols in MolecularBiology, (1987). Suitable vectors include the pEGF-N1 (Clontech) whichcontains the human cytomegalovirus (CMV) promoter, and the vector pcDNA.

Any cell may be used which is capable of expressing the transfectednucleotide sequences and in which an NLS facilitates transfer of atransmembrane protein away from the cell membrane. Suitable cellsinclude prokaryotic cells, including bacterial cells, and eukaryoticcells. Suitable eukaryotic cells include isolated mammalian cells, yeastcells, plant cells, insect cells, nematode cells and fungal cells.Suitable mammalian cells include human cell lines, rodent cell lines,hamster cell lines, non-human primate cell lines.

In one embodiment, the cell is transfected with a number of nucleotidesequences, each encoding a different NLS-containing transmembraneprotein and a different detectable moiety. Interference with traffickingof the transmembrane protein away from the cell membrane by a testcompound can be related to interaction of the compound with a particulartransmembrane protein by the identity of the detectable moiety whosemovement from the cell surface is interrupted.

In a further embodiment, for higher throughput initial screening, thecell is transfected with a greater number of nucleotide sequences, eachencoding a different NLS-containing transmembrane protein and adetectable moiety, some of the detectable moieties being common to morethan one transmembrane protein. If initial screening indicates that acandidate compound is interacting with one or more of the transmembraneproteins, the compound is rescreened using a cell expressing fewertransmembrane proteins, or only one, until the specific interactingtransmembrane protein is identified.

In cells transfected with more than one transmembrane protein, there maybe oligomerisation between pairs of proteins as discussed herein, andthis may affect the interpretation of the effect of a candidatecompound. Subsequent rescreening of the compound using cells transfectedwith only one transmembrane protein allows clarification of theinteraction of the compound with a particular protein.

Alternatively, for multiply transfected cells, transmembrane proteinsmay be selected which have been shown not to oligomerise with eachother.

Identification of Interacting Compounds

In one embodiment of the invention, nucleated cells are transfected witha nucleotide sequence encoding a protein comprising a transmembraneprotein containing an NLS and a detectable moiety and incubated for asuitable period of time to allow expression of the NLS-transmembraneprotein and commencement of its accumulation at the cell membrane. ForGPCRs and transporters, for example, a time period of about 6 to 24hours is suitable. One of skill in the art can readily determine asuitable incubation time for other transmembrane proteins by observationof the accumulation of the protein at the cell membrane. All of theexpressed transmembrane protein need not have reached the cell membranewhen the candidate compound is added. Test cells are then contacted witha candidate compound which is to be tested for interaction with thetransmembrane protein for a period of time which is sufficient to allowtranslocation of a substantial portion of the NLS-transmembrane protein,preferably at least 20%, more preferably at least 50%, and still morepreferably at least 90%, away from the cell membrane and into or towardsthe nucleus in a control cell not treated with compound.

Depending on the transmembrane protein, this period of time may be fromabout 6 hours to about 72 hours; a time period of about 24 to about 48hours is suitable for most transmembrane proteins examined. One of skillin the art can readily determine a suitable time by observation ofcontrol cells.

Test compounds are initially tested generally at a concentration ofabout 1 to 10 micromolar.

Test and control cells are then examined to determine the distributionof the detectable moiety and thereby the distribution of theNLS-transmembrane protein. The distribution of the detectable moiety maybe determined by various methods. For example, when the detectablemoiety is an optically detectable protein, the cells may be examined bydirect microscopy and the amount of protein in the nucleus comparedbetween test and control cells. In another embodiment, the amounts ofdetectable protein or peptide remaining in the membrane of control andtest cells are compared. In several microscopic fields (5-10), eachcontaining 30-100 cells, the location of the detectable moiety in thesecells is determined and counted for each location. The percentage ofcells having cell surface, or nuclear labelling and the sum of all thefields is then calculated for the treated and control cells.

In a further example, when the detectable moiety is an antigenicepitope, the cells are contacted with a detectable antibody systemcontaining an antibody specific for the epitope, as described above. Forexample, a first antibody specific for the epitope may be used, followedby a fluorescently labelled second antibody specific for the firstantibody, the fluorescent signal being quantified by fluorometer.

Where control cells show a substantial portion, preferably at least 50%,of the transmembrane protein translocated away from the cell membraneand test cells show retention of the transmembrane protein at the cellmembrane, relative to control cells, this indicates interaction of thetest compound with the transmembrane protein. In a preferred embodiment,interaction is indicated when the level of protein at the cell membraneis higher in the test cells by at least 10%, preferably by at least 15%,and more preferably by at least 20%.

The proportion of the detectable moiety remaining at the cell membraneon exposure to the interacting compound is related to the concentrationand potency of the compound. For example, the use of a known potent GPCRantagonist in micromolar concentration typically resulted in about 50 to100% of the protein remaining at the cell surface, 0 to 15% in thenucleus and the remainder in the cytoplasm. Lower nanomolarconcentrations of the same antagonist resulted in retention of 20 to 40%of the protein at the cell surface, with the rest of the protein in thecytoplasm and nucleus. In the untreated control cells, 0-15% of theprotein was detectable at the cell surface, with the remainder in thecytoplasm and nucleus.

In a variant of this method, used where the transmembrane protein hasknown ligands, an expressed NLS-containing transmembrane protein withouta detectable moiety is used and distribution of the protein in the cellafter treatment with a test compound is determined by isolation of thecell membrane fraction and determination of its transmembrane proteincomponent using detectably labelled ligand, as discussed above.

In a further embodiment of the invention, a similar method is used toidentify compounds which interact with an NLS-transmembrane protein topromote its translocation away from the cell surface and into or towardsthe nucleus. Cells are transfected and incubated to permit expression ofthe NLS-transmembrane protein and its accumulation at the cell surface.Preferably, the cells are incubated until at least about 70 to 90% ofthe expressed transmembrane protein has accumulated at the cell surface.For many transmembrane proteins, a time period of about 12 to about 24hours from transfection is suitable.

Test cells are then contacted with a candidate compound, and individualtest and control cells are immediately observed in real time for up to 4hours to observe the distribution of the detectable moiety. An increasedaccumulation of detectable moiety in the nucleus of test cells comparedwith control cells indicates that the test compound has promotedtranslocation of the transmembrane protein. In a preferred embodiment,interaction is indicated when test cells show nuclear accumulationincreased by at least 5%, preferably by at least 10%, and morepreferably by at least 20%.

A further embodiment of the invention is a method for identifyingcompounds which, although they do not themselves prevent translocationof an NLS-containing transmembrane protein away from the cell membrane,nevertheless can interfere with the interaction of the transmembraneprotein with an interacting compound.

Compounds which have proved negative in the first screening methoddescribed above may be tested by this further method for their abilityto compete with a known interacting compound.

In this method, cells are transfected as described above and incubatedfor a suitable period of time to allow expression and accumulation ofthe transmembrane protein at the cell surface, for example for about 24to about 48 hours.

Test cells and control cells are then contacted with a compound known tointeract with the transmembrane protein, either a known ligand or aninteracting compound identified by the method described above, for about24 to about 48 hours. Test cells are then contacted with a candidatecompound and test cells and control cells are observed after 1 hour, atone or more time points, up to 24 hours, to determine distribution ofthe NLS-transmembrane protein within the cells as described above. Incontrol cells, the known interacting compound causes the transmembraneprotein to be retained at the cell membrane. If the candidate compoundcompetes with the interacting compound, test cells show a reduction oftransmembrane protein at the cell surface and increased translocation ofthe protein away from the cell surface. In a preferred embodiment,interaction is indicated when test cells show a reduction of at least10%, preferably 15%, and more preferably 20%.

In a further embodiment, a cell which endogenously expresses anNLS-containing transmembrane protein may be employed, in conjunctionwith a first compound which has been demonstrated to interact with theprotein and inhibit its transfer from the cell membrane, thus retainingthe protein at the cell membrane. When such a system is contacted with acandidate compound, if that compound interacts with the transmembraneprotein and competes with the first compound, an increased transfer ofthe protein away from the cell membrane is observed.

Identifying Transmembrane Protein Interactions with Other Proteins

A number of transmembrane proteins, including GPCRs, transporters,tyrosine kinase receptors, the cytokine receptors for insulin,insulin-like growth factors, the epidermal growth factor and vascularendothelial growth factor, are capable of both homo- andheterooligomerisation (see, for example, review of GPCRs in George etal., 2002). As used herein, “oligomerisation” of a protein meansassociation of two or more molecules of the protein.

For hypothetical receptors A and B, the cell surface may contain dimersAA, BB and AB and it is believed that these may represent threedifferent functional complexes and therefore three different drugtargets. It is therefore important to identify which transmembraneproteins can interact with each other or with other proteins byoligomerisation.

In further embodiments, the invention provides methods for determiningwhether two transmembrane proteins are capable of oligomerisation orwhether a transmembrane protein and a non-transmembrane protein arecapable of oligomerisation.

In one embodiment, a nucleated cell is co-transfected with a firstnucleotide sequence encoding a first transmembrane protein containing anNLS and a second nucleotide sequence encoding a second transmembraneprotein lacking an NLS but carrying or linked to a detectable moiety.Creation of these nucleotide sequences is as described above. After asuitable time interval to allow for expression of the encoded proteins,accumulation at the cell membrane and subsequent translocation of theNLS-containing protein away from the cell membrane to or towards thenucleus, the distribution of the detectable moiety in the cell isdetermined, for example by determining an increase of detectable moietyin the nucleus or by a decrease of detectable moiety at the cellsurface.

It has been found that when cells are doubly transfected, and the firstand second transmembrane protein are the same, except that onetransmembrane protein contains an inserted NLS and the other does not,there is a slowing of the transfer of the NLS-containing transmembraneprotein to the cell nucleus compared with transfer in a cell transfectedonly with the NLS-containing protein. The process of proteintranslocation to the nucleus now may take about 24 to 48 hours. In thismethod, therefore, the cells are incubated for about 24 to 48 hoursbefore examination of the distribution of protein in the cell.

Translocation of the detectable moiety from the cell surface to ortowards the nucleus indicates that the first transmembrane protein hascarried the second transmembrane protein away from the cell surface,indicating oligomerisation of the first and second proteins. Retentionof the detectable moiety at the cell surface indicates a lack ofinteraction between the proteins.

When the first and second transmembrane proteins are the same protein,the method allows the identification of the ability of the protein tohomodimerise. When the first and second transmembrane proteins aredifferent, the method allows the identification of the ability of twodifferent proteins to heterodimerise and permits the determination ofthe specificity of interaction between two transmembrane proteins.

The method may be carried out either in the absence of ligand activationor in the presence of a ligand of either protein.

Using this method, oligomerisation has been demonstrated both within andbetween different classes of GPCRs and within and between other classesof transmembrane proteins.

In addition, interactions have been detected between GPCRs and non-GPCRtransmembrane proteins, for example between the D5 dopamine receptor andthe GABA-A receptor, and between transmembrane proteins andnon-transmembrane proteins.

The invention therefore generally provides a method for detectingoligomerisation between two proteins by the method described above,where a cell is co-transfected with one of the proteins containing anNLS and the other protein carrying a detectable signal.

Co-transfection of a cell with a first transmembrane protein containingan NLS and a second detectably labelled protein, such as a transmembraneprotein from a different group, which has been shown by the method ofthe invention to oligomerise with the first protein, provides a cellwhich can be used to screen candidate compounds for interaction witheither the first or second protein. A compound which interacts witheither protein will influence oligomerisation or translocation of theoligomerised proteins away from the cell membrane. Compounds whichinteract with one member of the protein pair or with the oligomer tocause retention of the detectable protein at the cell surface or tocause accelerated translocation of the detectable protein away from thecell surface may be identified by this method.

In a further embodiment, a cell which endogenously expresses anNLS-containing transmembrane protein is transfected with a nucleotidesequence encoding a second transmembrane protein carrying a detectablemoiety but lacking an NLS. Oligomerisation of the two proteins isindicated by trafficking of the detectable moiety away from the cellmembrane and into or towards the nucleus.

In a further embodiment, a membrane protein containing an NLS may beused to identify novel interacting proteins. In this method, anNLS-containing transmembrane protein is expressed in a cell and isallowed to translocate to the nucleus. The nuclei are then harvested andassayed for newly appeared protein bands by Coomassie staining or silverstaining and then identification by mass spectroscopy. The control willbe nuclei from cells expressing the membrane protein without a NLS.

Use of FRET for Detection of Nuclear Translocation.

In a further aspect of the invention, involving fluorescence resonanceenergy transfer (FRET) (Hailey et al., 2002), a nucleated cell isco-transfected with a first nucleotide sequence encoding a firstNLS-containing transmembrane protein linked to a first opticallydetectable protein and a second nucleotide sequence encoding a secondnon-NLS-containing transmembrane protein linked to a second opticallydetectable protein, whose fluorescence can be activated by the emissionof the first optically detectable protein when these are in closeproximity. For example, the first protein may be linked to GFP and thesecond any other optically detectable moiety that can be activated bythe laser activated emission spectrum of GFP. This second opticallydetectable moiety, after activation by the GFP, emits at a differentwavelength. Where oligomers are formed between the two transmembraneproteins, the two labels are in close proximity to each other and theirFRET interaction can be detected. The physical interaction is detectedby selective fluorescence activation of the donor and detection ofemission by the acceptor, using the FRET method or its variants such asphotobleaching FRET, FRAP or FLIM. Lack of a FRET interaction indicateslack of oligomerisation.

Confocal microscopy with FRET between two fluorescent molecules may beperformed (e.g. the spectral pairs GFP and DsRed2, or CFP and YFP) toobtain a quantifiable signal indicating translocation to the nucleus.FRET requires an overlap between the emission and excitation spectra ofdonor and acceptor molecules and a proximity of under 100 angstroms(10-100), making FRET a highly suitable system to assay for specificclose protein-protein interactions in cells. The fluorescent proteinslisted above are excellent spectral partners. A resident fluorophore inthe nucleus would enable FRET to occur when a transmembrane proteintagged with second fluorophore is translocated to the nucleus. This willfacilitate an easy readout, using a FRET plate reader. This method isuseful for detecting interactions between two transmembrane proteins orbetween a transmembrane protein and another protein and provides asignal readout more amenable to automation.

This method can also be used in GPCR agonist and antagonist screeningprocedures. In the antagonist screening method, a reduction of a FRETsignal between a GPCR-NLS-GFP trafficked to the nucleus with afluorophore in the nucleus of treated cells compared to non treatedcells would indicate an antagonist effect. In the agonist screeningmethod, the increase in the FRET signal between a GPCR-NLS-GFPtrafficked to the nucleus with a fluorophore in the nucleus of treatedcells compared to non treated cells would indicate an agonist effect. Ina further embodiment, the doubly transfected cells may be treated withan agonist before examination for evidence of oligomerisation, sincethis may be enhanced in the presence of agonist. In the measurement ofreceptor:receptor interactions, a GPCR-NLS-GFP is co-expressed with asecond GPCR-DsRED. If these receptors interact with each other andtraffic together to the nucleus a nuclear FRET signal will be detected.If the receptors do not interact then no FRET signal will be obtained inthe nucleus. FRET may also be measured between twofluorophore-conjugated antibodies recognising incorporated ot nativeepitopes on the GPCRs.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of chemistry, molecular biology, protein and peptidebiochemistry and immunology referred to but not explicitly described inthis disclosure and examples are reported in the scientific literatureand are well known to those skilled in the art.

Materials and Methods

Green fluorescent protein: a DNA sequence encoding the Aequoria victoriagreen fluorescent protein (Prasher et al., 1992) was obtained fromClontech, U.S.A.

Red fluorescent protein: a DNA sequence encoding the red fluorescentproteins (Matz et al., 1999) pDsRed2 and pDsRed2-nuc were obtained fromClontech, U.S.A. This construct encodes a protein derived from Discosomasp.

COS cells and HEK cells were obtained from American Type CultureCollection, Washington, D.C. The cell culture media were prepared bylaboratory services at the University of Toronto.

Antagonist and agonist compounds were obtained from various commercialsources such as Sigma Chemical Company U.S.A.

Antibodies used for immunodetection of epitope tags were obtained fromthe following sources: Anti-HA monoclonal antibody was obtained fromRoche Diagnostics, U.S.A. Anti-FLAG monoclonal antibody was obtainedfrom Sigma Chemical Company, U.S.A. Anti-c-myc monoclonal antibody wasobtained from Santa Cruz, U.S.A.

Radioligand ³H-SCH 23390 used in the receptor binding assay was obtainedfrom NEN Perkin Elmer, U.S.A.

Creation of DNA Constructs

Nucleotide sequences encoding GPCR's or transporters were obtained fromthe Genbank (http://www.ncbi.nlm.nih.gov:80/entrez) web site,established by the National Library of Science. A nucleotide sequenceencoding a selected transmembrane protein was attached to a nucleotidesequence encoding a selected detectable signal protein. The constructswere cloned into the vector system, pEGFP (Clontech) or the pDsRed2-N1vector or the vector pcDNA3.

1a. Construction of the Human D1 Dopamine Receptor with a NLS in theProximal Carboxyl Tail (Helix 8) and Fused to GFP (D1-GFP andD1-NLS-GFP).

Using the PCR method with the following experimental conditions, DNAencoding the human D1 dopamine receptor in the vector pcDNA3, wassubjected to PCR. The reaction mixture contained water (32 microlitres),10×Pfu buffer (Stratagene) (5 microlitres), dNTP (2′-deoxynucleoside5′-triphosphate, 10 mM) (5 microlitres), DMSO (5 microlitres),oligonucleotide primers (100 ng) (1 microlitre each), DNA template (100ng), Pfu enzyme (5 units). Total volume was 50 microlitres. Thefollowing PCR conditions were used, one cycle at 94° C. for 2 mins,30-35 cycles at 94° C. for 30 secs, 55° C. for 30 secs, 72° C. for 1min, per cycle, and then one cycle at 72° C. for 5 mins.

Primer set for amplification of the DNA encoding the D1-dopaminereceptor:

HD1-P1: (SEQ ID NO:1)5′ GAGGACTCTGAACACCGAATTCGCCGCCATGGACGGGACTGGGCTGGT G 3′ HD1-P2: (SEQ IDNO:2) 5′ GTGTGGCAGGATTCATCTGGGTACCGCGGTTGGGTGCTGACCGTT 3′

The restriction site EcoR1 was incorporated in the primer HD1-P1, andrestriction site Kpn1 was incorporated into the primer HD1-P2. The PCRproduct, which contained no stop codon was unidirectionally subclonedinto vector pEGFP (from Clontech) at EcoR1 and Kpn1 and inframe with thestart codon of the GFP protein.

The NLS sequence, KKFKR (SEQ ID NO: 157) from the human AT1 receptor wasinserted into DNA encoding the base of TM7 (helix 8) of the D1 dopaminereceptor by PCR, replacing the natural sequence coding for DFRKA.

The primer set for the construction of DNA encoding D1-NLS:

HD1-NLSF: (SEQ ID NO:3)5′ CCTAAGAGGGTTGAAAATCTTTTAAATTTTTTAGCATTAAAGGCATAA ATG 3′ HD1-NLSR:(SEQ ID NO:4) 5′ GCCTTTAATGCTAAAAAATTTAAAAGATTTTCAACCCTCTTAGGATG C 3′

Using the DNA encoding D1-GFP as template, PCR with the primers HD1-P1and HD1-NLSF resulted in a product of 1000 bp (PCR#1). Using DNAencoding D1-GFP PCR with primers HD1-P2 and HD1-NLSR resulted in aproduct of 300 bp (PCR#2). A subsequent PCR carried out with HD1-P1 andHD1-P2 primers resulted in a product of 1300 bp using the product fromPCR#1 and the product from PCR#2 as templates. The resulting DNAencoding D1-NLS was subcloned into vector PEGFP at EcoR1 and Kpn1restriction sites.

All the additional constructs described below were made using the samePCR method and experimental conditions as described above for the D1dopamine receptor, but with specific primers as described below.

1b. Constructing the Human Dopamine D1 Receptor Containing a NLS andFused to RFP (D1-NLS-RFP)

The NLS sequence K K F K R was inserted into the helix 8 segment of theintracellular carboxyl tail of the human D1 receptor by PCR method asfollows. Using the DNA encoding the human D1 in pcDNA3 vector astemplate, the first PCR was carried out with HD1-P1 and HD1-NLSR primersresulting in a 1 kb product. A second PCR was done using HD1-P2 andHD1-NLSF primers resulting in a 300 bp product. Using PCR#1 and PCR#2products as templates, the final PCR was done with HD1-P1 and HD1-P2primers which generated a 1.3 kp product.

D1NLS was subcloned into vector pDsRed (Clontech) at EcoRI and KpnI andfused to RFP.

Primer Sequences:

HD1-P1: (SEQ ID NO:1) 5′ GAGGACTCTGAACACCGAATTCGCCGCCATGGACGGGACTGGGCTGGTG 3′ HD1-P2: (SEQ ID NO:2)5′ GTGTGGCAGGATTCATCTGGGTACCGCGGTTGGGTGCTGACCGTT 3′ HD1-NLSF: (SEQ IDNO:4) 5′ GCCTTTAATGCTAAAAAATTTAAAAGATTTTCAACCCTCTTAGGATG C 3′ HD1-NLSR:(SEQ ID NO:3) 5′ CCTAAGAGGGTTGAAAATCTTTTAAATTTTTTAGCATTAAAGGCATAA ATG 3′D1-wildtype: (SEQ ID NO:5) N P I I Y A F N A D F R K A F S T L LD1NLS-Helix8: (SEQ ID NO:6) N P I I Y A F N A K K F K R F S T L L

1c. Construction of the Dopamine D1 Receptor with a Hemagglutinin (HA)Epitope Tag in the Amino Terminus

The HA-Tag is as follows:

Nucleotide sequence: (SEQ ID NO:7) TACCCTTACGACGTGCCGGATTACGCC HA aminoacid sequence: (SEQ ID NO:8) Y P Y D V P D Y A

The HA epitope tag was inserted into the amino terminal of the human D1receptor using D1-pcDNA3 as template with the following primers:

P1HA-BamH: (SEQ ID NO:9)5′ggatccactagtaacggccgccagaccaccATGGGATACCCGTACGACGTCCCCGACTACGCAAGGACTCTGAACACCTCTGCC 3′ P2-NotI: (SEQ ID NO:10)5′ ggccgccagctgcgagTTCAGGTTGGGTGCTGACCG 3′The resulting amplified cDNA (1.3 kb) was subcloned into pcDNA3 vectorat BamH I and Not I.

D1 wildtype: (SEQ ID NO:11) M R T L N T S A M D G T G L V V D1-HA tag:(SEQ ID NO:12) M G Y P Y D V P D Y A R T L N T S A M D G T G L V V

1d. Construction of the Human Dopamine D1 Receptor with a HA Epitope andNLS in the Proximal Carboxyl Tail (Helix 8) (D1 HA-NLS)

Primer set for the PCR amplification of DNA encoding D1-NLS (helix 8),using DNA encoding D1-HA as template. Using DNA D1-HA as template withprimers T7 and HD1-NLSR primers the resulting amplified DNA was 1000 bp(PCR#1). Using DNA D1-HA as template with primers Sp6 and HD1-NLSRprimers the resulting DNA was 300 bp, (PCR#2). Using primers T7 and Sp6primers and the product of PCR#1 and PCR#2 as templates the resultingDNA was 1300 bp (PCR#3).

HD1-NLSR: (SEQ ID NO:3)5′ CCTAAGAGGGTTGAAAATCTTTTAAATTTTTTAGCATTAAAGGCAT AAATG 3′ HD1-NLSF:(SEQ ID NO:4) 5′ GCCTTTAATGCTAAAAAATTTAAAAGATTTTCAACCCTCTTAGGAT GC 3′

The result D1 HA-NLS (helix 8) PCR was blunt-ended into pcDNA3 at EcorV.The correct orientation clone was sequenced.

D1-HA wildtype: (SEQ ID NO:5) N P I I Y A F N A D F R K A F S T L LD1HA-NLS (helix 8): (SEQ ID NO:6) N P I I Y A F N A K K F K R F S T L L

1e. Construction the Dopamine D1 Receptor with a NLS in IntracellularLoop 3, Fused to GFP (D1-NLS-IC3-GFP)

Primer set for the construction of D1-NLS-IC3-GFP:

D1NLSF-IC3: (SEQ ID NO:13) 5′ GGAAAGTTCTTTTAAGAAGAAGTTCAAAAGAGAAAC 3′D1-NLSR-IC3: (SEQ ID NO:14) 5′ GTTTCTCTTTTGAACTTCTTCTTAAAAGAACTTTCC 3′

Using D1 pcDNA3 template:

PCR#1: HD1-P1 and D1NLSR-IC3 primers

PCR#2: HD1-P2 and D1NLSF-IC3 primers (500 bp)

PCR#3: HD1-P1 and HD1-P2 primers using PCR#1 and PCR#2 as templates (1.3kb)

The resulting DNA fragment encoding D1-NLS-IC3 was subcloned into vectorpEGFP at EcoR1 and Kpn1.

D1-wildtype: (SEQ ID NO:15) Q P E S S F K M S F K R E T K V LD1-NLS-IC3: (SEQ ID NO:16) Q P E S S F K K K F K R E T K V L

The NLS sequence KKFKR was inserted into the IC loop 3 segment of the D1receptor replacing the sequence MFSKR, using D1 pcDNA3 as template.

Using the DNA encoding D1 in pcDNA3 as template, PCR was carried outwith the following primers HD1-P1 and D1-NLSR-IC3 resulting in a productof 800 bp (PCR#1). Using DNA encoding D1 in pcDNA3 with primers HD1-P2and HD1-NLSF-IC3 resulted in a product of 500 bp (PCR#2). A subsequentPCR carried out with HD1-P1 and HD1-P2 primers resulted in a product of1300 bp using the product from PCR#1 and the product from PCR#2 astemplates. The resulting construct encoding D1-NLS was subcloned intovector pEGFP at EcoR1 and Kpn1 restriction sites.

1f. Construction of Human D1 Dopamine Receptor with a NLS inIntracellular Loop 2 fused with GFP (D1-NLS-IC2-GFP)

The primer set for the construction of DNA encoding D1NLS-IC2

D1NLSF-IC2: (SEQ ID NO:17) 5′ CCGGTATGAGAAAAAGTTTAAACGCAAGGCAGCCTTC 3′D1-NLSR-IC2: (SEQ ID NO:18) 5′ GGCTGCCTTGCGTTTAAACTTTTTCTCATACCGGAAAGG3′

Using DNA encoding D1 dopamine receptor in pcDNA3 as template, PCR withthe primers HD1-P1 and D1NLSR-IC2 (PCR#1), resulted in a product of 500bp. Using DNA encoding D1 dopamine receptor in pcDNA3 as a template withprimers HD1-P2 and D1NLSF-IC2 (PCR#2) resulted in a product of 800 bp. Asubsequent PCR carried out with primers HD1-P1 and HD1-P2 using PCR#1and PCR#2 as templates resulted in a product of 1300 bp.

The resulting DNA encoding D1NLS-IC2 PCR was subcloned into vector EGFPat EcoR1 and Kpn1.

D1-wildtype: (SEQ ID NO:19) N P F R Y E R K M T P K A A F I L ID1-NLS-IC2: (SEQ ID NO:20) N P F R Y E K K F K R K A A F I L I

1g. Construction of the Human D1 Dopamine Receptor with a NLS inIntracellular Loop 1 Fused with GFP (D1-NLS-IC1-GFP)

The primer set for the construction of DNA encoding D1-NLS-IC1.

D1-NLSF-IC1: (SEQ ID NO:21) 5′ GTGCTGCCGTTAAAAAGTTCAAACGCCTGCGGTCCAAGG3′ D1-NLSR-IC1: (SEQ ID NO:22)5′ GGACCGCAGGCGTTTGAACTTTTTAACGGCAGCACAGACC 3′

Using the DNA encoding D1 dopamine receptor in pcDNA3 as template, PCRwith the primers HD1-P1 and D1-NLSR-IC1 (PCR#1), resulted in a productof 300 bp. Using DNA encoding D1 dopamine receptor in pcDNA3 as templatePCR with primers HD1-P2 and D1NLSF-IC1 resulted in a product of 1000 bp(PCR#2). A subsequent PCR carried out with primers HD1-P1 and HD1-P2using PCR#1 and PCR#2 as templates resulted in a product of 1300 bp.

The resulting DNA encoding D1-NLS-IC1 was subcloned into vector PEGFP atEcoR1 and Kpn1.

D1-wildtype: (SEQ ID NO:23) L V C A A V I R F R H L R S K V T ND1-NLS-IC1: (SEQ ID NO:24) L V C A A V K K F K R L R S K V T N

1h. Construction of Human Dopamine D1 Receptor with an Alternate NLS inthe Proximal Carboxyl Tail and Fused to GFP (D1-NLS2-GFP)

The PCR method was used to introduce the NLS sequence PKKKRKV (SEQ IDNO: 129) in replacement of the natural sequence ADFRKAF in the D1receptor. The DNA encoding the D1 dopamine receptor in pcDNA3 wassubjected to PCR with the primers HD1-P1 and HD1-NLS2R, resulting in aproduct of 1 kb (PCR#1). Another PCR using D1 in pcDNA3 with primersHD1-P2 and HD1-NLS2F resulted in a product of 300 bp (PCR#2). The thirdPCR using PCR#1 and PCR#2 as templates HD1-P1 and HD1-P2 primersresulted in a product of 1.3 kb, and was subcloned into vector PEGFP atEcoR1 and Kpn1.

HD1-NLS2F: (SEQ ID NO:25)5′ GCCTTTAATCCTAAAAAAAAAAGAAAGGTTTCAACCCTCTTAGG 3′ HD1-NLS2R: (SEQ IDNO:26) 5′ CCTAAGAGGGTTGAAACCTTTCTTTTTTTTTTAGGATTAAAGGC 3′ D1-wildtype:(SEQ ID NO:27) N P I I Y A F N A D F R K A F S T L L D1-NLS2: (SEQ IDNO:28) N P I I Y A F N P K K K R K V S T L L

2. Construction the Dopamine D2 and D2-NLS Dopamine Receptors Fused toGFP (D2-GFP and D2-NLS-GFP)

Primer set for amplification of the DNA in pcDNA3 encoding theD2-dopamine receptor.

HD2-P1: (SEQ ID NO:29) 5′ GGCCGTGGCTCCACCGAATTCGCCGCCATGGATCCACTGAATCTG3′ HD2-P2: (SEQ ID NO:30) 5′ CTGTGCGGGCAGGCAGGGTACCGCGCAGTGGAGGATCTTCAGG3′

The restriction site EcoR1 was incorporated into primer HD2-P1, and therestriction site Kpn1 was incorporated into primer HD2-P2. The D2-PCRproduct, which contained no stop codon, was unidirectionally subclonedinto vector pEGFP (Clontech) at EcoR1 and Kpn1 and inframe with thestart codon of the GFP protein.

Primer set for the construction of D2-NLS-GFP

HD2-NLSF: (SEQ ID NO:31) 5′ CACCACCTTCAACAAAAAATTCAAAAGAGCCTTCCTGAAGATCC3′ HD2-NLSR: (SEQ ID NO:32)5′ GGATCTTCAGGAAGGCTCTTTTGAATTTTTTGTTGAAGGTGGTG 3′

The NLS sequence KKFKR was inserted into the base of TM7 segment of theD2 receptor replacing the sequence IEFRK, using D2-GFP DNA construct astemplate.

Using the DNA encoding D2-GFP as template, PCR was carried out with thefollowing primers HD2-P1 and HD2-NLSR resulting in a product of 1300 bp(PCR#1). Using DNA encoding D2-GFP PCR with primers HD2-P2 and HD1-NLSFresulted in a product of 100 bp (PCR#2). A subsequent PCR carried outwith HD2-P1 and HD2-P2 primers resulted in a product of 1400 bp usingthe product from PCR#1 and the product from PCR#2 as templates. Theresulting construct encoding D2-NLS was subcloned into vector pEGFP atEcoR1 and Kpn1 restriction sites.

3. Construction of DNA Encoding the D3 and D5 Dopamine Receptors Fusedto GFP (D3-GFP and D5-GFP)

Primer set for amplification of the DNA in pcDNA3 encoding theD3-dopamine receptor.

HD3-Hind: (SEQ ID NO:33)5′ GGCATCACGCACCTCAAGCTTGCCGCCATGGCATCTCTGAGTCAGC 3′ HD3-Kpn: (SEQ IDNO:34) 5′ GAGTGTTCCCTCTTCTGCGGTACCGCGCAAGACAGGATCTTGAGG 3′

The restriction site HindIII was incorporated into primer HD3-Hind, andthe restriction site KpnI and was incorporated into primer HD3-Kpn. TheD3-PCR product, which contained no stop codon, was unidirectionallysubcloned into vector pEGFP at HindIII and KpnI and inframe with thestart codon of the GFP protein.

Primer set for amplification of the DNA in pcDNA3 encoding the D5dopamine receptor.

T7: (SEQ ID NO:35) 5′ AATACGACTCACTATAG 3′ HD5-Kpn: (SEQ ID NO:36)5′ CGCCAGTGTGATGGATAATGGTACCGCATGGAATCCATTCGGGGTG 3′

The restriction site KpnI and was incorporated into primer HD5-Kpn. TheD5-PCR product, which contained no stop codon, was unidirectionallysubcloned into vector pEGFP at EcoRI and KpnI and inframe with the startcodon of the GFP protein.

4. Construction of the Histamine1 and Histamine1-NLS Receptors Fused toGFP (H1-GFP and H1-NLS-GFP)

Primer set for amplification of the DNA, from human genomic DNA,encoding the encoding the H1 histamine receptor.

H1-MET: (SEQ ID NO:37) 5′ GCGCCAATGAGCCTCCCCAATTCC 3′ H1-STOP: (SEQ IDNO:38) 5′ GAGCCTCCCTTAGGAGCGAATATGC 3′

This H1-PCR product was used as a template for the subsequent PCRexperiment.

Primer set for amplification of the DNA encoding the H1-GFP construct.

H1-PST: (SEQ ID NO:39) 5′ CGCCTGCAGGCCGCCATGAGCCTCCCCAATTCCTCC 3′H1-APA: (SEQ ID NO:40) 5′ CCGGTGGATCCCGGGCCCCGGAGCGAATATGCAG 3′

The restriction site PstI was incorporated into primer H1-PST, and therestriction site ApaI was incorporated into primer H1-APA. This H1-PCRproduct, which contained no stop codon, was unidirectionally subclonedinto vector PEGFP at PstI and ApaI and inframe with the start codon ofthe GFP protein.

Primer set for amplification of the DNA encoding the H1-NLS-GFP

H1-NLSR: (SEQ ID NO:41)5′ GGGCCCCGGAGCGAATATGCAGAATTCTCTTGAATGTCCTCTTGAATT TTTTATTGCACAAGG 3′

The NLS sequence: KKFKR was inserted into the DNA encoding the TM7segment of the H1 receptor by the PCR method, using the H1-GFP template,replacing the sequence ENFKK. PCR with H1-PST and H1-NLSR primers gave aproduct of 1500 bp. The resulting fragment encoding H1-NLS was subclonedinto vector PEGFP at PstI and ApaI restriction sites.

5. Construction the Cysteinyl Leukotriene Receptor 1 and CysLT1-NLSFused to GFP (CysLT1-GFP and CysLT1-NLS-GFP).

Primer set for amplification of the DNA in pcDNA3 encoding the CysLT1receptor.

LT1-EcorI: (SEQ ID NO:42) 5′ AAGAATTCGCCACCATGGATGAAACAGGAAATCTG 3′LT1-KpnI: (SEQ ID NO:43) 5′ GGGTACCGCTACTTTACATATTTCTTCTCC 3′

The restriction site EcoR1 was incorporated into primer LT1-EcoRI andthe restriction site Kpn1 was incorporated into primer LT1-KpnI. TheCysLT1-PCR DNA product, which contained no stop codon, wasunidirectionally subcloned into vector PGFP at EcoR1 and Kpn1 andinframe with the start codon of the GFP protein.

Primer set for amplification of the DNA encoding the CysLT1-NLS-GFP

LT1-NLSF: (SEQ ID NO:44) 5′ TTCTTTTCTGGGAAAAAATTTAAGAGAAGGCTGTCTAC 3′LT1-NLSR: (SEQ ID NO:45) 5′ TGTAGACAGCCTTCTCTTAAATTTTTTCCCAGAAAAG 3′

The NLS sequence KKFKR was inserted into the DNA encoding the TM7segment of the CysLT1 by PCR, using DNA encoding the CysLT1-GFP astemplate, replacing the sequence GNFRK. Using the DNA encodingCysLT1-GFP as template, PCR with the following primers LT1-EcoRI andLT1-NLSR resulted in a fragment of 900 bp (PCR#1). Using DNA encodingCysLT1-GFP PCR with primers LT1-KpnI and LT1-NLSF resulted in a fragmentof 100 bp (PCR#2). A subsequent PCR carried out with LT1-EcoRI andLT1-KpnI primers resulted in a product of 1000 bp using the product fromPCR#1 and the product from PCR#2 as templates. The resulting DNAencoding CysLT1-NLS was subcloned into vector PEGFP at EcoR1 and Kpn1restriction sites.

6. Construction of the Cysteinyl Leukotriene Receptor CysLT2 andCysLT2-NLS Fused to GFP (CysLT2-GFP and CysLT2-NLS-GFP)

Primer set for amplification of the DNA in pcDNA3 encoding the CysLT2receptor.

LT2-EcoRI: (SEQ ID NO:46)5′ CTTTTTGTGTCTGTTTCTGAATTCGCCACCATGGAGAGAAAATTTAT G 3′ LT2-KpnI: (SEQID NO:47) 5′ GAACAGGTCTCATCTAAGAGGTACCGCTACTCTTGTTTCCTTTCTC 3′

The restriction site EcoR1 was incorporated into primer LT2-EcoRI, andthe restriction site Kpn1 was incorporated into primer LT2-KpnI. TheCysLT2 product, which contained no stop codon, was unidirectionallysubcloned into vector pEGFP at EcoR1 and Kpn1 and inframe with the startcodon of the GFP protein.

Primer set for the amplification of the CysLT2-NLS-GFP

LT2-NLSF: (SEQ ID NO:48) 5′ GCTGGGAAAAAATTTAAAAGAAGACTAAAGTCTGCAC 3′LT2-NLSR: (SEQ ID NO:49) 5′ GTCTTCTTTTAAATTTTTTCCCAGCAAAGTAATAGAGC 3′

The NLS sequence KKFKR was inserted into the TM7 segment of the CysLT2by PCR method replacing the sequence ENFKD. Using the DNA encodingCysLT2-EGFP as template, a PCR with the following primers LT2-EcoR1 andLT2-NLSR primers resulted in a fragment of 900 bp (PCR#1). Using DNAencoding LT2-KpnI and LT2-NLSF primers a PCR resulted in a fragment of200 bp (PCR#2). A subsequent PCR carried out with LT2-EcoR1 and LT2-KpnIprimers using the product of PCR#1 and the product of PCR#2 as templatesresulted in a product of 1100 bp. The resulting DNA encoding CysLT2-NLSwas subcloned into vector pEGFP at EcoR1 and Kpn1 restriction sites.

7. Construction of the M1 Muscarinic Receptor and the Muscarinic NLSReceptor Fused to GFP (M1-GFP and M1-NLS-GFP)

Primer set for amplification of the DNA encoding the muscarinic receptor(M1) from human genomic DNA.

M1-MET: (SEQ ID NO:50) 5′ CCCCACCTAGCCACCATGAACACTTC 3′ M1-STOP: (SEQ IDNO:51) 5′ GGGGACTATCAGCATTGGCGGGAGG 3′

Primer set for MR1-EGFP

M1-PST: (SEQ ID NO:52) 5′ CCCCACCTGCAGCCACCATGAACACTTCAGCC 3′ M1-BAMH:(SEQ ID NO:53) 5′ GGGGAGGATCCGCGCATTGGCGGGAGGGAGTGC 3′

The restriction site PstI was incorporated into primer M1-PST, and therestriction site BamHI was incorporated into primer M1-BAMH. The M1 PCRproduct, which contained no stop codon, was unidirectionally subclonedinto vector PEGFP at PstI and BamHI and inframe with the start codon ofthe EGFP protein.

Primer set for M1-NLS EGFP

M1-NLSF: (SEQ ID NO:54) 5′ CGCACTCTGCAACAAAAAATTCAAACGCACCTTTCGCC 3′M1-NLSR: (SEQ ID NO:55) 5′ GGCGAAAGGTGCGTTTGAATTTTTTGTTGCAGAGTGCG 3′

The NLS sequence KKFKR was inserted into the TM7 segment of the M1 byPCR, using the MR1 template, replacing the sequence KAFRD. Using the DNAencoding MR1 as template, PCR with the following primers using M1-PSTand M1-NLSR resulted in a product of 1200 bp (PCR#1). Using DNA encodingMR1 a PCR with primers reaction using M1-BAMH and M1-NLSF primersresulted in a product of 100 bp (PCR#2). A subsequent PCR carried outwith M1-PST and ML-BAMH primers resulted in a product of 1300 bp usingthe product from PCR#1 and the product from PCR#2 as templates. Thisfragment encoding MR1-NLS was subcloned into vector PEGFP at PstI andBamHI restriction sites.

8. Construction of the Serotonin (5HT1B) and the Serotonin NLS ReceptorsFused to GFP (5HT1B-GFP and 5HT1B-NLS-GFP)

Primer set for amplification of the DNA encoding the 5HT1B receptor fromthe plasmid pcDNA3 encoding the 5HT1B receptor.

5HT1B-E1: (SEQ ID NO:56) 5′ GGGGCGAATTCGCCGCCATGGAGGAACCGGGTGC 3′5HT1B-KPN: (SEQ ID NO:57) 5′ GCAAACGGTACCGCACTTGTGCACTTAAAACGTA 3′

The restriction site EcoR1 was incorporated into primer 5HT1B-E1 and therestriction site Kpn1 was incorporated into primer 5HT1B-KPN. The5HT1B-PCR product, which contained no stop codon, was unidirectionallysubcloned into vector PEGFP at EcoR1 and Kpn1 and inframe with the startcodon of the GFP protein.

Primer set for 5HT1B-NLS EGFP

5HT1B-NLSF: (SEQ ID NO:58) 5′ ATGTCCAATAAAAAATTTAAAAGAGCATTCCATAAACTG 3′5HT1B-NLSR: (SEQ ID NO:59) 5′ GGAATGCTCTTTTAAATTTTTTATTGGACATGGTATAG 3′

The NLS sequence: KKFKR was inserted into the TM7 segment of the 5HT1Bby PCR using 5HT1B-EGFP template, replacing the sequence EDFKQ. Usingthe DNA encoding 5HT1B-EGFP as template, a PCR with the followingprimers with 5HT1B-Eland HD1-NLSF primers gave a product of 1100 bp(PCR#1). Using DNA encoding 5HT1B-EGFP with 5HT1B-KPN and HD1-NLSRprimers resulted in a product of 100 bp (PCR#2). A subsequent PCRcarried out with 5HT1B-E1 and 5HT1B-KPN primers resulted in a product of1200 bp using the product from PCR#1 and the product from PCR#2 astemplates. The resulting DNA encoding 5HT1B-NLS was subcloned intovector PEGFP at EcoR1 and Kpn1 restriction sites.

9. Construction of the beta2-adrenergic (beta2-AR) and the beta2-AR-NLS1Receptors Fused to GFP (beta2-AR-GFP and beta2AR-NLS1-GFP)

Primer set for amplification of the DNA encoding the beta2-AR receptorfrom pcDNA3.

T7: (SEQ ID NO:60) 5′ AATACGACTCACTATAG 3′ Beta2-Kpn: (SEQ ID NO:61)5′ GCCGCCAGTGTGATGGATACTGGTACCGCTAGCAGTGAGTCATTT GTAC 3′

The restriction site Kpn1 was incorporated into primer beta2-Kpn. Thebeta 2-AR product, which contained no stop codon, was unidirectionallysubcloned into vector pEGFP at EcoR1 and Kpn1 and inframe with the startcodon of the GFP protein.

The NLS sequence KKFKR was inserted into the TM7 segment of the beta2-ARby PCR using beta2-AR-EGFP template, replacing the sequence PDFRI. Usingthe DNA encoding beta2-AR-EGFP as template, a PCR with the followingprimers with T7 and B2-NLSR primers resulted in a product of 1100 bp(PCR#1). Using DNA encoding beta2-AR-EGFP with beta2-Kpn and B2-NLSFprimers resulted in a product of 300 bp (PCR#2). A subsequent PCRcarried out with primers T7 and beta2-Kpn resulted in a product of 1300bp using the product from PCR#1 and the product from PCR#2 as templates.The resulting DNA encoding beta2-NLS was subcloned into vector pEGFP atEcoR1 and Kpn1 restriction sites.

10. Construction of the beta 2-adrenergic Receptor with an Alternate NLSand Fused to GFP (beta2-NLS2-GFP)

Primer set for amplification of the DNA encoding the beta2-NLS2 receptorfrom pcDNA3.

B2D1NLSF: (SEQ ID NO:62) 5′ CCCCTTATCTACGCCTTTAGCGCAAAGAAGTTCAAGCGC 3′B2D1NLSR: (SEQ ID NO:63) 5′ GCGCTTGAACTTCTTTGCGCTAAAGGCGTAGATAAGGGG 3′

Using the DNA encoding beta2-AR-GFP as template, a PCR with thefollowing primers with T7 and B2D1NLSR primers resulted in a product of1000 bp (PCR#1). Using DNA encoding beta2-AR-GFP with beta2-Kpn andB2D1NLSF primers resulted in a product of 300 bp (PCR#2). A subsequentPCR carried out with primers T7 and beta2-Kpn primers using PCR#1 andPCR#2 as templates resulted in a product of 1300 bp. The resulting DNAencoding beta2-NLS2 was subcloned into vector pEGFP at EcoR1 and Kpn1restriction sites.

The NLS sequence AFSAKKFKR (SEQ ID NO: 158) was inserted into the TM7segment of the beta2-AR by PCR using beta2-GFP template, replacing thesequence CRSPDFRIA.

The resulting DNA encoding beta2-NLS2 was subcloned into vector pEGFP atEcoR1 and Kpn1 restriction sites.

11. Construction of the beta 2-adrenergic Receptor with an Alternate NLSand Fused to GFP (beta2-NLS3-GFP)

The NLS sequence K K F K R (SEQ ID NO: 129) was inserted into anotherlocation of the proximal segment of the carboxyl tail of the beta2-AR.Using the DNA encoding the beta2AR in pcDNA3 vector as template, PCR wascarried out with T7 and r B2-NLS3R primers resulting a 1000 bp product.PCR using primers Beta2-Kpn and B2-NLS3F resulted in a 300 bp product.Using PCR#1 and PCR#2 products as templates PCR with T7 and Beta2-Kpnprimers generated a 1300 bp product (beta2AR-NLS3) which was subclonedinto vector PEGFP at EcoR1 and Kpn1.

Primer set for Beta2-NLS3-GFP

B2-NLS3F: (SEQ ID NO:64) 5′ CTGCCGGAGCAAAAAATTCAAAAGAGCCTTCCAGGAGC 3′B2-NLS3R: (SEQ ID NO:65) 5′ CCTGGAAGGCTCTTTTGAATTTTTTGCTCCGGCAGTAG 3′Wildtype Beta2: (SEQ ID NO:66) N P L I Y C R S P D F I R A F Q E L LBeta2AR-NLS3: (SEQ ID NO:67) N P L I Y C R S K K F K R A F Q E L L

12. Construction of the Dopamine Transporter Fused to GFP (DAT-GFP)

The full length cDNA encoding the human dopamine transporter (hDAT) wasamplified using DAT in pcDNA3 as template by PCR with primer T7 andprimer DT-1 (5′ CGTCTCTGCTCCCTGGTACCGCCACCTTGAGCCAGTGG 3′: SEQ ID NO:68). This PCR product contained no stop codon and was unidirectionallysubcloned into vector pEGFP (from Clontech) at the EcoR1 and Kpn1restriction sites and inframe with the start codon of the GFP protein.

13a. Construction of the Human Dopamine Transporter Containing a NLS andFused to RFP (DAT-NLS-RFP)

The cDNA encoding the human dopamine transporter (hDAT) was amplified byPCR with 1718 and hDAT-NLSF primers, producing a fragment of 100 bp. ThecDNA encoding the human dopamine transporter (hDAT) was also amplifiedby PCR with T7 and hDAT-NLSR primers, producing a fragment of 1.7 kB.These two PCR fragments were used as templates with T7 and 1718 primers,resulting in a fragment of 1.8 kB.

Primer T7: (SEQ ID NO:69) 5′ TAATACGACTCACTATAGGG 3′ Primer 1718: (SEQID NO:70) 5′ CGTCTCTGCTCCCTGGTACCGCCACCTTGAGCCAGTGG 3′ hDAT-NLSF: (SEQID NO:71) 5′ CTATGCGGCCAAAAAGTTCAAAAGACTGCCTGGGTCC 3′ hDAT-NLSR: (SEQ IDNO:72) 5′ CAGGCAGTCTTTTGAACTTTTTGGCCGCATAGATGGGC 3′

This PCR product was unidirectionally subcloned into vector pRFP atEcoR1 and Kpn1 and inframe with the start codon of the RFP protein.

The resulting PCR fragment encoded the NLS sequence K K F K R after TM12as follows:

DAT-wild type: (SEQ ID NO:73) S S M A M V P I Y A A Y K F C S L P G S FR E K DAT-NLS: (SEQ ID NO:74) S S M A M V P I Y A A K K F K R L P G S FR E K

13b. Construction of the Human Dopamine Transporter with a NLS and Fusedto GFP (DAT-NLS-GFP)

The NLS sequence K K F K R was inserted into the proximal carboxyl tailfollowing the transmembrane 12 segment of the human DAT. Using the DNAencoding the human DAT-cDNA in pcDNA3, as template, the first PCR wascarried out with T7 and hDAT-NLSR primers resulting a 1.7 kb product. Asecond PCR was done using 1718 and hDAT-NLSF primers resulting in a 100bp product, then using PCR#1 and PCR#2 products as templates the finalPCR was done with T7 and 1718 primers which generated a 1.8 kp product(DAT-NLS) which was subcloned into vector pEGFP (Clontech) at EcoR1 andKpn1 and fused GFP.

Sequences of the Primers:

hDAT-NLSF: (SEQ ID NO:75) 5′ CTATGCGGCCAAAAAGTTCAAAAGACTGCCTGGGTCC 3′hDAT-NLSR: (SEQ ID NO:76) 5′ CAGGCAGTCTTTTGAACTTTTTGGCCGCATAGATGGGC 3′Human DAT wildtype: (SEQ ID NO:77) S S M A M V P I Y A A Y K F C S L P GS F R E K Human DAT-NLS: (SEQ ID NO:78) S S M A M V P I Y A A K K F K RL P G S F R E K

14. Construction of the Human Serotonin Transporter Fused to GFP(SERT-GFP)

The full length human SERT cDNA was isolated by PCR from pcDNA3containing the SERT cDNA, using the two following primers:

SERT-HIND: (SEQ ID NO:79) 5′ GTCATTTACTAAGCTTGCCACCATGGAGACGACGCCCTTG 3′SERT-KPN: (SEQ ID NO:80) 5′ CCTCTCGGTGAGTGGTACCGCCACAGCATTCAAGCGG 3′

This PCR product contained no stop codon and was unidirectionallysubcloned into vector pEGFP (Clontech) at HindIII and KpnI and inframewith the start codon of the GFP protein.

15. Construction of the Human Low Density Lipoprotein Receptor Fused toGFP (LDL-R-GFP)

The full length cDNA encoding LDL was subjected to PCR with LDLR-HINDand LDLR-KPN primers:

LDLR-HIND: (SEQ ID NO:81) 5′ GGACACTGCCTGGCAAAGCTTGCGAGCATGGGGCCCTGG 3′LDLR-KPN: (SEQ ID NO:82) 5′ GGCGGGACTCCAGGCAGGTACCGCCGCCACGTCATCCTCC 3′

This PCR product (2600 bp) contained no stop codon and wasunidirectionally subcloned into vector pEGFP (Clontech) at HindIII andKpnI and inframe with the start codon of the GFP protein.

16. Construction of the Human Low Density Lipoprotein Receptor with aNLS and Fused to GFP (LDLR-NLS-GFP)

The NLS sequence KKFKR was inserted into DNA encoding the LDL receptorby PCR, replacing the natural sequence coding for RLKNI.

The primer set for the construction of DNA encoding LDL-NLS:

LDL-NLSF: (SEQ ID NO:83) 5′ CTATGGAAGAACTGGAAAAAATTTAAAAGAAACAGCATCAAC3′ LDL-NLSR: (SEQ ID NO:84)5′ CAAAGTTGATGCTGTTTCTTTTAAATTTTTTCCAGTTCTTCC 3′

Using the human DNA encoding LDL cDNA in pcDV1 as template, PCR with theprimers LDLR-HIND and LDL-NLSR resulted in a product of 2450 bp (PCR#1).Using DNA encoding LDL as a template with primers LDLR-KPN and LDL-NLSFresulted in a product of 150 bp (PCR#2). A subsequent PCR carried outwith primers LDLR-HIND and LDLR-KPN using the product of PCR#1 and PCR#2as template resulted in a product of 2600 bp.

The resulting PCR contained the NLS sequence K K F K R mutation asfollows:

Human LDL-R wildtype: (SEQ ID NO:85) F L L W K N W R L K N I N S I N F DN P Human LDL-R: (SEQ ID NO:86) F L L W K N W K K F K R N S I N F D N P

This PCR product contained no stop codon and was unidirectionallysubcloned into vector PEGFP (Clontech) at HindIII and KpnI and inframewith the start codon of the GFP protein.

17. Construction of the Epidermal Growth Factor Receptor Fused to GFP(EGFR-GFP)

The full length human EGFR cDNA in Prkf vector was isolated by PCR withthe two following primers:

HER-XHO: (SEQ ID NO:87) 5′ GCTCTTCGGGCTCGAGCAGCGATGCGACCCTCCGGGACGG 3′HER-KPN: (SEQ ID NO:88) 5′ CTATCCTCCGTGGTACCGCTGCTCCAATAAATTCACTGC 3′

This PCR product (3600 bp) contained no stop codon and wasunidirectionally subcloned into vector pEGFP (Clontech) at XhoI and KpnIand inframe with the start codon of the GFP protein.

18. Construction of the Human Serotonin Transporter with a NLS and Fusedto GFP (SERT-NLS-GFP)

The NLS sequence KKFKR was inserted into DNA encoding the SERT by PCR,replacing the natural sequence coding for GTFKE.

The primer set for the amplification of the DNA encoding SERT-NLS.

SERT-NLSF: (SEQ ID NO:89) 5′ GATCATCACTCCAAAGAAATTTAAAAGACGTATTATT 3′SERT-NLSR: (SEQ ID NO:90) 5′ TAATACGTCTTTTAAATTTCTTTGGAGTGATGATCAACCG 3′

Using the human SERT-cDNA in PcDNA3 as template, PCR with the primersSERT-HIND and SERT-NLSR resulted in a product of 1800 bp (PCR#1). UsingDNA encoding SERT as a template with primers SERT-KPN and SERT-NLSFresulted in a product of 100 bp (PCR#2). A subsequent PCR carried outwith primers SERT-HIND and SERT-KPN primers using the product of PCR#1and PCR#2 as template resulted in a product of 1900 bp.

The resulting PCR product encoded the NLS sequence K K F K R mutationafter TM12 of the SERT as follows:

Human SERT wildtype: (SEQ ID NO:91) R L I I T P G T F K E R I I K S I THuman SERT: (SEQ ID NO:92) R L I I T P K K F K R R I I K S I T

This PCR product contained no stop codon and was unidirectionallysubcloned into vector pEGFP (Clontech) at HindIII and KpnI and inframewith the start codon of the GFP protein.

19. Construction of the Metabotropic glutamate-4-receptor Fused to GFP,with and without NLS (mGluR4-GFP and mGluR4-NLS-GFP)

The DNA encoding mGluR4 was isolated from a rat cDNA using the primerset

GLUR4-HIND: (SEQ ID NO:93) 5′ GGGTCTCTAAGCTTGCCGCCATGTCCGGGAAGGG 3′GLUR4-ECORI: (SEQ ID NO:94) 5′ CCGCGGCCCGGAATTCGGATGGCATGGTTGGTG 3′

A restriction site HindIII was incorporated into primer GLUR4-HIND, anda restriction site EcoRI was incorporated into primer GLUR4-ECORI. ThemGluR4-PCR product, which contained no stop codon, was unidirectionallysubcloned into vector EGFP (Clontech) at HindIII and EcorI and inframewith the start codon of the GFP protein.

The NLS KKFKR was introduced into the DNA encoding the mGluR4 replacingthe natural sequence KRKRS.

Primer set for the amplification of the DNA to introduce the NLS intothe Rat mGluR4-EGFP

GLUR4-NLSF: (SEQ ID NO:95) 5′ CGTGCCCAAGAAATTCAAGCGCCTCAAAGCCGTGGTC 3′GLUR4-NLSR: (SEQ ID NO:96) 5′ CGGCTTTGAGGCGCTTGAATTTCTTGGGCACGTTCTGC 3′

Using the rat DNA encoding GluR4 as template PCR with the primersGLUR4-HIND and GLUR4-NLSR resulted in a product of 2600 bp (PCR#1).Using DNA encoding GluR4 with primers GLUR4-ECORI and GLUR4-NLSFresulted in a product of 160 bp (PCR#2). A subsequent PCR carried outusing the product of PCR#1 and PCR#2 as template, with primers:GLUR4-HIND and GLUR4-ECORI, resulted in a product of 2760 bp.

The resulting PCR contained the NLS sequence K K F K R mutation asfollows:

Rat mGluR4 Wildtype: (SEQ ID NO:97) F H P E Q N V P K R K R S L K A V VT A A T Rat mGluR4: (SEQ ID NO:98) F H P E Q N V P K K F K R L K A V V TA A T

This PCR product was unidirectionally subcloned into vector pEGFP(Clontech) at HindIII and EcoRI and inframe with the start codon of theGFP protein.

20. Construction of the Human Insulin Receptor Fused to GFP (IR-GFP)

The full length IR cDNA in plasmid pRK5 was isolated with the two PCRprimers:

HIR-HIND: (SEQ ID NO:99) 5′ GGAGACCCCAAGCTTCCGCAGCCATGGGCACCGGGGGCC 3′HIR-APA: (SEQ ID NO:100) 5′ CCCCGCCACGGGCCCCGGAAGGATTGGACCGAGGCAAGG 3′The PCR product (4.2 kb) contained no stop codon and wasunidirectionally subcloned into vector pEGFP (Clontech) at HindIII andApaI and fused to the GFP protein.

21. Construction of the Human Insulin Receptor with a NLS and Fused toGFP (IR-NLS-GFP)

The NLS sequence KKFKR was introduced into the human insulin receptor toreplace the sequence LYASS.

Using the human insulin receptor cDNA in pRK5 vector as template, thefirst PCR #1 with HIR-HIND and HIR-NLSR primers generated a 2.9 kbproduct, the second PCR #2 with HIR-APA and HIR-NLSF primers generated a1.3 kb product, and then using the products from PCR#1 and PCR #2 astemplates, the third PCR #3 produced a fragment with HIR-HIND andHIR-APA primers (4.2 kb). This contained no stop codon and wasunidirectionally subcloned into vector PEGFP at HindIII and ApaI andthus fused to the GFP protein.

Primers for HIR-NLS:

HIR-NLSF: (SEQ ID NO:101) 5′ CCGCTGGGACCGAAAAAATTTAAGAGAAACCCTGAGTATCTC3′ HIR-NLSR: (SEQ ID NO:102)5′ GATACTCAGGGTTTCTCTTAAATTTTTTCGGTCCCAGCGGCCC 3′

22. Construction of the Human Erythropoietin Receptor Fused to GFP(EPO-GFP).

Using PCR method and the cDNA in pc3.1 vector encoding the humanErythropoietin receptor (EPO) as template, the full length cDNA wasisolated with the following primers:

T7: (SEQ ID NO:103) 5′ TAATACGACTCACTATAGGG 3′ EPO-KPN: (SEQ ID NO:104)5′ GACTGCAGCCTGGTGGTACCGCAGAGCAAGCCACATAGCTGGGG 3′This PCR product (1.6 kb) contained no stop codon and wasunidirectionally subcloned into vector pEGFP at HindIII and KpnI andfused to the GFP protein.

23. Construction of the Human Erythropoietin Receptor with a NLS andFused to GFP (EPO-NLS-GFP).

The NLS sequence KKFKR was inserted into the DNA encoding the EPOreceptor by PCR, replacing its natural sequence RRALK.

Using the human EPO-cDNA in pc3.1 as template, the first PCR #1 with T7and EPO-NLSR primers generated a 900 bp product, the second PCR #2 withEPO-KPN and EPO-NLSF primers generated a 700 bp product, and then usingthe products from PCR#1 and PCR #2 as templates, the third PCR #3 withT7 and EPO-KPN primers produced a 1.6 kb fragment. This PCR product (1.6kb) contained no stop codon and was unidirectionally subcloned intovector pEGFP at HindIII and KpnI and thus fused to the GFP protein.

Primer Sequences:

T7: (SEQ ID NO:105) 5′ TAATACGACTCACTATAGGG 3′ EPO-KPN: (SEQ ID NO:106)5′ GACTGCAGCCTGGTGGTACCGCAGAGCAAGCCACATAGCTGGGG 3′ EPO-NLSF: (SEQ IDNO:107) 5′ GCTGCTCTCCCACAAAAAGTTTAAGCGGCAGAAGATCTGG 3′ EPO-NLSR: (SEQ IDNO:108) 5′ CCAGATCTTCTGCCGCTTAAACTTTTTGTGGGAGAGCAGC 3′ Human EPOwildtype: (SEQ ID NO:109) T V L A L L S H R R A L K O K I W P G I PHuman EPO NLS: (SEQ ID NO:110) T V L A L L S H K K F K R O K I W P G I P

24. Construction of the Human Epidermal Growth Factor Receptor Fused toGFP (EGFR-GFP)

Using the human epidermal growth factor receptor cDNA in Prk5 vector astemplate, the full length cDNA was isolated by PCR with the twofollowing primers:

HER-XHO (SEQ ID NO:111) (5′ GCTCTTCGGGCTCGAGCAGCGATGCGACCCTCCGGGACGG 3′)and HER-KPN (SEQ ID NO:112) (5′ CTATCCTCCGTGGTACCGCTGCTCCAATAAATTCACTGC3′)This PCR product (3.6 kb) contained no stop codon and wasunidirectionally subcloned into vector pEGFP (Clontech) at XhoI and KpnIand fused to the GFP protein.

25. Construction of the Human Epidermal Growth Factor Receptor with anNLS and Fused to GFP (EGFR-NLS-GFP)

The NLS sequence K K F K R was inserted into the sequence of the humanepidermal growth factor receptor by PCR method as follows. Using thehuman EGFR cDNA in Prk5 vector as template, the first PCR was carriedout with HER-XHO and EGF-NLSR primers resulting in a 2.1 kb product. Asecond PCR was done using HER-KPN and EGF-NLSF primers resulting a 1.5kb product, and then using PCR#1 and PCR#2 products as templates, thefinal PCR was done with HER-XHO and HER-KPN primers, which generated a3.6 kp product (EGFR-NLS) which was subcloned into vector pEGFP(Clontech) at XhoI and KpnI and fused to GFP.

Primer Sequences:

EGF-NLSF: (SEQ ID NO:113) 5′ CACATCGTTCGGAAGAAGTTTAAGCGGAGGCTGCTGC 3′EGF-NLSR: (SEQ ID NO:114) 5′ CCTGCAGCAGCCTCCGCTTAAACTTCTTCCGAACGATGTG 3′Human EGFR wildtype: (SEQ ID NO:115) R R R H I V R K R T L R R L L Q E RE Human EGFR-NLS: (SEQ ID NO:116) R R R H I V R K K F K R R L L Q E R E

26. Construction of the Human D1 Dopamine Receptor Containing 2 NLSs andFused to RFP (D1-NLS(Helix 8 and C-Tail)-RFP)

A second NLS sequence K K K R K was inserted into the carboxyl tailsegment of the human D1-NLS-Helix 8 by PCR method as follows. Using theDNA encoding the human D1-NLS-Helix 8 in pDsRed vector as template, thefirst PCR was carried out with HD1-P1 and HD1-NLSCR primers resulting ina 1.2 kb product, and a second PCR was done using HD1-P2 and HD1-NLSCFprimers resulting in a 100 bp product. Then using PCR#1 and PCR#2products as templates, the final PCR was done with HD1-P1 and HD1-P2primers which generated a 1.3 kp product (D1-NLS-Helix 8 and C-tail)which was subcloned into pDsRed vector at EcoRI and KpnI and fused tothe DsRed protein.

Primer Sequences:

HD1-P1: (SEQ ID NO:117) 5′ GAGGACTCTGAACACCGAATTCGCCGCCATGGACGGGACTGGGCTGGTG 3′ HD1-P2: (SEQ ID NO:118)5′ GTGTGGCAGGATTCATCTGGGTACCGCGGTTGGGTGC TGACCGTT 3′ HD1-NLSCF: (SEQ IDNO:119) 5′ CCTCTGAGGACCTGAAAAAGAAGAGAAAGGCTGGCATCGCC 3′ HD1-NLSCR: (SEQID NO:120) 5′ GGCGATGCCAGCCTTTCTCTTCTTTTTCAGGTCCTCAGAGG 3′ D1-wildtype:(SEQ ID NO:121) N P I I Y A F N A D F R K A F S T L L . . . S S E D L KK E E A A G I A D1-NLS (Helix 8 and C-tail): (SEQ ID NO:122) N P I I Y AF N A K K F K R F S T L L . . . S S E D L K K K R K A G I A

27. Construction of the Mu opioid Receptor Fused to GFP (Mu-GFP)

Using the DNA encoding the Mu opioid receptor in pcDNA3 vector astemplate, PCR was carried out with the following two primers.

RATMU1: (SEQ ID NO:123) 5′ CCTAGTCCGCAGCAGGCCGAATTCGCCACCATGGACAGCAGCACC3′ RATMU-2: (SEQ ID NO:124)5′ GATGGTGTGAGACCGGTACCGCGGGCAATGGAGCAGTTTCTGCC 3′Restriction site EcoRI was incorporated into primer RATMU-1. Restrictionsite KpnI was incorporated into primer RATMU-2

The PCR product (1.2 kb) which contained no stop codon, was thenunidirectionally subcloned into vector pEGFP (Clontech) at EcoR1 andKpn1 and thus fused to GFP.

28. Construction of the Mu opioid Receptor Containing a NLS and Fused toGFP (Mu-NLS-GFP)

The NLS sequence K K F K R (SEQ ID NO: 157) was inserted into theproximal carboxyl tail segment (helix 8) of the Mu opioid receptor byPCR as follows. Using the DNA encoding the Rat Mu in pcDNA3 as template,the first PCR was carried out with RATMU1 and MU-NLSR primers resultinga 1000 bp product, another second PCR was done using RATMU-2 and MU-NLSFprimers resulting a 200 bp product. Using PCR#1 and PCR#2 products astemplates the final PCR was done with RATMU1 and RATMU2 primersgenerated a 1200 bp product (Mu-NLS) which was subcloned into vectorpEGFP at EcoR1 and Kpn1 and fused to GFP.

Primer Sequences:

RATMU-1: (SEQ ID NO:125)5′ CCTAGTCCGCAGCAGGCCGAATTCGCCACCATGGACAGCAGCACC 3′ RATMU-2: (SEQ IDNO:126) 5′ GGATGGTGTGAGACCGGTACCGCGGGCAATGGAGCAGTTTCTGCC 3′ MU-NLSF:(SEQ ID NO:127) 5′ GCCTTCCTGGATAAAAAATTCAAGCGATGC 3′ MU-NLSR: (SEQ IDNO:128) 5′ GCATCGCTTGAATTTTTTATCCAGGAAGGCG 3′

Cell Culture and Transfection

COS-7 monkey kidney cells and HEK293T human embryonic kidney cells(American Type Culture Collection, Manassa, Va.) were maintained asmonolayer cultures at 37° C. and 5% CO₂ in minimal essential mediumsupplemented with 10% fetal bovine serum and antibiotics. For cellmembrane harvesting, 100 mm plates of cells were transiently transfectedat 70-80% confluency using lipofectamine reagent (Life Technologies,Rockville, Md.). For confocal microscopy studies, 60 mm plates of cellswere transiently transfected at 10-20% confluency using lipofectaminereagent. Six hours after transfection, the solution was removed andfresh media added and again replaced with fresh media 24 hours aftertransfection.

Transfection medium was prepared by mixing 120 microlitres mediumwithout antibiotics and/or fetal bovine serum (FBS) and 15 microlitresslipofectamine in a 14 ml tube. 2 micrograms DNA construct encoding thedesired fusion protein and 120 microlitres medium were mixed and addedto the 14 ml tube, which was mixed gently and incubated at roomtemperature for 25 minutes. A further 4 ml of medium was added andmixed. If multiple transmembrane proteins are being transfected, thecDNAs are mixed and transfected together. Growth medium was removed froma plate of cells and replaced with the transfection mixture from the 14ml tube. Cells were incubated with the transfection mixture for 5-6hours, after which the mixture was removed and replaced with regulargrowth medium containing FBS and antibiotics. Cells were incubated witha change of regular growth medium on the second day.

Treatment with Test Compounds

Protocol for Determining Retardation of Translocation Off Cell Surface

Test compounds were prepared in a stock solution of 1 millimolarconcentration and diluted in growth medium to achieve a finalconcentration ranging between 10 nanomolar and 10 micromolar when addedto cell plates. Fresh compound-containing medium was added to cells at 6hours, 22 hours, 30 hours and 42 hours after transfection.

Protocol for Determining Promotion of Translocation Off Cell Surface

Test compounds were prepared in a stock solution of 1 millimolar anddiluted in growth medium at 37° C. to achieve a final concentration of10 micromolar when added to cells. Cell cultures were examinedmicroscopically to focus on a single cell and to detect the presence ofsurface expression of the detectable label protein. Growth medium wasreplaced with compound-containing medium and the cells were examinedmicroscopically in real time 5, 10, 15, 20, 30 and 35 mins. afteraddition of compound for changes in distribution of the detectablemoiety.

Microscopy

Cells were visualised using the LSM510 Zeiss confocal laser microscope.GFP was visualised following excitation with the argon laser at 488 nmexcitation wavelength and the DsRed was visualised following excitationwith the helium neon laser at 543 nm wavelength for excitation. Theconfocal images were captured on disk and evaluated. In each experiment,multiple fields of cells (n=6-8 with 30-90 cells each) were counted andevaluated for localisation of the signal on the cell surface, in thecytoplasm and in the nucleus.

Fluorocytometry

A 96 well plate was coated with poly-L-ornithine ( 1/10 in PBS) andincubated for one hour. 50,000 cells were added to each well andtransfected with cDNA encoding the epitope-tagged receptor, usingLipofectamine (Invitrogen, U.S.A.). The medium (MEM) was changed every12 hours and contained test drug at varying concentrations or vehicle.48 hours later, cells were washed and fixed with 4% paraformaldehyde andincubated for 30 min on ice. Cells were then incubated with the primaryantibody directed against the epitope and then with the secondaryantibody conjugated to FITC (fluorescein isothiocyanate) and keptshielded from light. Excess antibody washed off and the signal detectedby reading the plate in a Cytofluor 4000 (PerSpective Biosystems,U.S.A.). The FITC was activated using light at 488 nm for excitation andthe signal read at its emission wavelength 530 nm.

Radioligand Binding

Cells were transfected with DNA encoding D1-NLS and treated with varyingconcentrations of antagonist drug or left untreated. After 48 hours, thecells were washed, harvested, lysed and homogenised by polytron. Themembrane fraction was collected by centrifugation and then layered over35% sucrose solution and centrifuged at 30,000 rpm at 4 degrees C. for90 min to collect the heavy membrane fraction. The supernatant was againcentrifuged at 35,000 rpm at 4 degrees C. for 60 min to collect thelight membrane fraction. The membranes were subjected to radioligandbinding assay using [³H]-SCH23390 with (+)butaclamol 10 micromolar usedto define specific binding. The incubation was at room temperature for 2hours, followed by rapid filtration and quantitation by scintillationcounting.

Isolation of Nuclei from Cultured Cells

Wash cells with PBS 3 times, using 10 ml and scrape gently off theculture dishes. Pool and spin the cells at 500 g for 5 min, at 4 degreesC. Resuspend pelleted cells in lysis buffer (Tris HCl 10 mM, pH 7.4,NaCl 10 mM, MgCl2 3 mM) and inhibitor cocktail (0.5% leupeptin, 1%soybean trypsin, 1% benzamidine) at a density of 50 million cells perml. Homogenize with sterile glass Teflon pestle B (tight clearance 20-50mm; Bellco Glass) using 100 up and down strokes.

Spin at 4 degrees C. for 700 g for 10 min, then sequentially centrifugesupernatant at 10 000 g, for 15 min at 4 C (to remove mitochondria) and120 000 g for 60 min, 4 degrees C. (to remove plasma membrane).

The nuclear pellet is resuspended in lysis buffer (with inhibitors) and0.1% of NP-40, kept on ice 5 min and then centrifuged at 700 g for 10min at 4 degrees C. The supernatant is discarded and washing processrepeated 3× with 15 ml lysis buffer. Nuclear pellet is resuspended in 2ml lysis buffer and loaded on top of discontinuous sucrose gradient madeby successive layering of 4.5 ml of 2.0 and 1.6 M sucrose containingMgCl2 1 mM and spun at 100 000 g for 60 min at 4 degrees C. The pelletat the bottom of the tube is collected and will contain pure nuclei.

Example 1 Dopamine D1 Receptor Fused to the Red Fluorescent Protein(D1-RFP) or Containing a NLS and Fused to the Red Fluorescent Protein(D1-NLS-RFP)

The sequence of the dopamine D1 receptor, which does not contain an NLS,was modified to replace the amino acids DFRKA at the base of TM7 domainwith the NLS sequence KKFKR (which corresponds to the NLS of the humanAT1 receptor), as described in the methods (see FIG. 1). DNA constructswere created encoding the D1 dopamine receptor fusion proteins D1-RFPand D1-NLS-RFP. COS cells were transfected with DNA encoding D1-NLS-RFPor D1-RFP (2 micrograms) and incubated for 24 and 48 hours. The cellswere examined by confocal microscopy at 100× magnification. Cells werecounted manually in 8 to 10 microscopic fields and the percentagelabelling in different subcellular compartments was calculated.

At 24 and 48 hours, cells transfected with D1-RFP showed expression atthe cell surface in the majority of cells, whereas cells transfectedwith D1-NLS-RFP showed little receptor expression at the cell surface,with nuclear localisation in 60% of the cells at 24 hours, and in 80% ofthe cells at 48 hours.

Example 2 Dopamine D1 Receptor Fusion Protein Containing a NLS(D1-NLS-RFP) Treated with Antagonist

COS cells were transfected with a construct encoding D1-NLS-RFP and withwildtype D1 (2 micrograms) for 48 hours. At 6, 22, 30 and 42 hours aftertransfection, the cells were treated with the dopamine D1 receptorantagonist SCH23390 (final concentration 10 micromolar). Also at 6, 22,30 and 42 hours after transfection, cells were treated with theantagonist (+)butaclamol (final concentration 10 μM). Control cellsreceived no antagonist treatment.

At 48 hours, the majority of control cells had detectable D1-NLS-RFP inthe nucleus. In contrast, the majority of antagonist-treated cells hadfluorescence only on the cell surface, while 42% had fluorescence bothon the surface and in the nucleus.

Example 3 Dopamine D1 Receptor (D1-GFP) Co-Expressed with D1 ReceptorContaining a NLS (D1-NLS)

HEK cells were transfected with DNA constructs encoding D1-NLS (3micrograms) and/or D1-GFP (1.5 micrograms), and incubated for 48 hours.The cells were also transfected with a plasmid encoding DsRed-NUC toverify the localisation of the nucleus (1 microgram).

Cells were also transfected with DNA encoding D1-GFP (2 micrograms),incubated for 48 hours and examined by confocal microscopy.

D1-GFP expressed alone revealed that 90% of the cells demonstrated cellsurface labelling and 10% showed both nuclear and cell surfacelabelling. With any DNA encoding a GPCR transfection, up to 10% of cellsmay be observed with a nuclear localisation.

Cells expressing D1-GFP and D1-NLS showed 35% of cells with both nuclearand cell surface labelling and 70% with receptor-expression on cellsurface only. This experiment indicated that D1-GFP co-trafficked withD1-NLS resulting from oligomerisation of the D1-NLS and D1-GFP.

Example 4 Dopamine D1 Receptor Containing a NLS (D1-NLS-GFP), wasTreated with an Antagonist in a Dose Response Study

HEK cells were transfected with DNA encoding D1-NLS-GFP (2 micrograms),and D1-WT (6 micrograms) for 48 hours. These cells were treated 6 hrsafter transfection with SCH-23390 (10 micromolar), or (+)butaclamol (10micromolar). The medium containing antagonist was changed at 6, 22, 30and 42 hours after transfection. Control cells received no antagonisttreatment.

Following SCH-23390 treatment for 48 hours, 58% of the cells had cellsurface expression of D1-NLS-GFP, less than 10% of the cells hadreceptor expression in the nucleus and 32% of the cells had receptorexpression on both the cell surface and in the nucleus.

Following (+)butaclamol treatment for 48 hours, 62% of the cells hadcell surface expression of the D1-NLS-GFP receptor, 10% had receptorexpression in the nucleus, and 28% of the cells had receptor expressionon the cell surface and in the nucleus.

Control cells at 48 hours showed approximately 65% with D1-NLS-GFPreceptor expression in the nucleus, and 35% with receptor expression inthe cytoplasm. No receptor D1-NLS-GFP expression was found on the cellsurface of control cells.

Incorporation of an NLS into the receptor sequence caused a veryefficient removal of the D1-NLS-GFP receptor from the cell surface andlocalisation in the nucleus.

Similar studies were carried out at various doses of SCH-23390 or(+)butaclamol. Results are shown in Tables 2 and 3.32% to 35% of controlcells showed receptor in cytoplasm.

TABLE 2 % cells receptor on SCH-23390 receptor on surface and inreceptor in concn surface nucleus nucleus 10 μM 58% 32% <10%  5 μM 46%42% 12% 1 μM 39% 46% 15% 0.5 μM 36% 44% 20% 0.2 μM 32% 49% 19% 0.0 μM 0% 62%-70%

TABLE 3 % cells receptor on (+)butaclamol receptor on surface and inreceptor in concn. surface nucleus nucleus 10 μM 62% 28% 10% 5 μM 47%43% 10% 1 μM 41% 43% 16% 0.5 μM 40% 41% 19% 0.2 μM 39% 21% 40% 0.0 μM 0% 62%-70%

Incorporation of an NLS into the receptor sequence caused a veryefficient removal of the D1 dopamine receptor from the cell surface andlocalisation in the nucleus. Treatment with D1 selective antagonistsprevented this receptor translocation in a dose-responsive manner.

Example 4a Expression of the Dopamine D1 Receptor with an Inserted NLS(D1-NLS-GFP) and Treatment with Agonists

HEK cells were transfected with a DNA construct encoding D1-NLS-GFP (1.5micrograms), and incubated with the D1 agonist SKF-81297 (10 micromolar)for 48 hours. At 6, 22, 30 and 42 hours after transfection, the cellswere treated with fresh medium containing SKF-81297 (final concentration10 micromolar). The cells were examined by confocal microscopy.

HEK cells were transfected with a DNA construct encoding D1-NLS-GFP (1.5micrograms of DNA), and incubated with the agonist pergolide (10micromolar) for 48 hours. At 6, 22, 30 and 42 hours after transfection,the cells were treated with fresh medium containing SKF-81297 (finalconcentration 10 micromolar). The cells were examined by confocalmicroscopy.

Control HEK cells were transfected with a DNA construct encodingD1-NLS-GFP (1.5 micrograms of DNA) and left untreated.

In the untreated cells after 48 hrs, there was no receptor detected atthe cell surface. With cells treated with SKF-81297, 59% of cells hadreceptor expression at the cell surface. With cells treated withperglolide there was receptor surface expression in 59% of the cells.Thus long-term treatment with agonists prevented the modified D1receptor from trafficking to the nucleus.

Example 5 Dopamine D1 Receptor with an Incorporated NLS (D1-NLS-RFP)Co-Expressed with the Wild Type D1 Receptor

COS cells were co-transfected with a DNA construct encoding D1-NLS-RFP(1 microgram) and a DNA sequence encoding the native dopamine D1receptor (D1-WT, 7 microgram) and incubated for 24 or 48 hours.

At 24 hours, DL-NLS-RFP was detected only at the cell surface, whereasat 48 hours, 80% of cells had D1-NLS-RFP in the nucleus. The wild typereceptor retarded the movement of the D1-NLS-RFP to the nucleus byhomo-oligomerisation.

Example 6 D1 Dopamine Receptor with an Incorporated NLS (D1-NLS-RFP)Co-Expressed with DL-GFP

COS cells were transfected with a construct encoding D1-NLS-RFP (4micrograms) and the dopamine D1-GFP (4 micrograms), and incubated for 48hours. The cells were examined by confocal microscopy.

D1-GFP was detected at the cell surface and a yellow fluoresence wasdetected in the nuclei, the latter indicating co-localisation of bothD1-NLS-RFP and D1-GFP in the nucleus, confirming oligomerisation ofD1-NLS-RFP and DL-GFP, leading to importation of D1-GFP into thenucleus.

Example 6a Expression of the D1 Dopamine Receptor with an NLS Insertedin the Third Intracellular Cytoplasmic Loop (D1-IC3-NLS-GFP)

HEK cells were transfected with a DNA construct encoding D1-IC3-NLS-GFP(2 micrograms), and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

In cells transfected with D1-IC3-NLS-GFP, the receptor was detected inthe nucleus of 85% of cells. Thus insertion of a NLS into the thirdintracellular loop enabled receptor trafficking to the nucleus.

Example 6b Expression of the D1 Dopamine Receptor with an NLS Insertedin the First Intracellular Cytoplasmic Loop (D1-IC1-NLS-GFP)

HEK cells were transfected with a DNA construct encoding D1-IC1-NLS-GFP(2 micrograms), and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

In cells transfected with D1-IC1-NLS-GFP, the receptor was detected inthe nucleus of 85% of cells. Thus insertion of a NLS into the firstintracellular loop enabled receptor trafficking to the nucleus.

Example 6c Effect of the Antagonist Butaclamol or SCH-23390 on theTrafficking of the D1 Dopamine Receptor with an NLS Inserted in theFirst Cytoplasmic Loop (D1-IC1-NLS-GFP)

HEK cells were transfected with a DNA construct encoding D1-IC1-NLS-GFP(2 micrograms), and treated with either butaclamol (final concentration1 micromolar or SCH-23390 (1 micromolar), for 48 hours. Nuclei werevisualised with DsRED-NUC (2 micrograms). The cells were examined byconfocal microscopy.

With the butaclamol treated cells, 82% had receptor on the cell surfaceor in the cytoplasm. 18% of the cells had receptor in the nucleus. Thustreatment with butaclamol reduced D1-IC1-NLS-GFP trafficking to thenucleus.

With the SCH-23390 treated cells, 77% of the cells had receptor on thecells surface or in the cytoplasm. 23% of the cells had receptor in thenucleus. Thus treatment with SCH-23390 reduced receptor trafficking tothe nucleus.

With the untreated cells 76% had receptor expression in the nucleus andcytoplasm.

Example 6d Effect of the Antagonist SCH-23390 on the Trafficking of theD1 Dopamine Receptor with an NLS Inserted in the Third Cytoplasmic Loop(D1-IC3-NLS-GFP)

HEK cells were transfected with a DNA construct encoding D1-IC3-NLS-GFP(2 micrograms), and treated with four different concentrations ofSCH-23390 (10 micromolar, 1 micromolar, 500 nanomolar and 100nanomolar), for 48 hours. Nuclei were visualised with DsRED-NUC (2micrograms). The cells were examined by confocal microscopy.

86% of the cells transfected with D1-IC3-NLS-GFP had the receptor in thenucleus, and 0% had receptor on the surface. With the SCH-23390 treatedcells, 84% had receptor in the nucleus and 15% of the cells had receptoron the surface. The insertion of an NLS at this position in the GPCRwill translocate the receptor to the nucleus efficiently but does notrespond to the drug.

Example 6e Expression of the D1 Dopamine Receptor with an NLS Insertedin the Second Intracellular Cytoplasmic Loop (D1-IC2-NLS-GFP)

HEK cells were transfected with a DNA construct encoding D1-IC2-NLS-GFP(2 micrograms), and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

In cells transfected with D1-IC2-NLS-GFP, the receptor was detected inthe nucleus of 51% of cells.

Example 6f Ability of the Dopamine D1 Receptor to Homodimerise, withStaggered Transfection

HEK cells were transfected with a DNA construct encoding D1-RFP (2micrograms) and after 24 hours incubation, the cells were transfectedwith a second DNA construct encoding D1-NLS-GFP (2 micrograms). Controlcells were transfected with the D1-RFP (2 micrograms) construct alone.The cells were incubated for 48 hours following the second transfectionand examined by confocal microscopy.

90% of the cells transfected with D1-RFP alone expressed receptor on thecell surface, and 6% of the cells expressed receptor in the nucleus. Incontrast, 97% of the cells expressing both forms of the receptorexpressed both receptors (red plus green equals yellow fluorescence) inthe nucleus. Thus, the D1 receptor without the NLS interacted with theD1 receptor with the NLS in order to traffic to the nucleus.

Example 7 Dopamine D5 Receptor (D5-GFP)

A construct encoding the dopamine D5 receptor-GFP (D5-GFP) was preparedand used to transfect COS cells (4 micrograms).

Cells transfected with dopamine D5-GFP, at 48 hours, showed mainly acytoplasmic localisation of receptor, with cell surface localisation inonly a few cells and no instances of nuclear localisation.

Example 8 Dopamine D1 with an Incorporated NLS (D1-NLS) Co-Expressedwith the D5 Dopamine Receptor (D5-GFP)

HEK cells were transfected with two DNA constructs, one encoding D1-NLS(7 micrograms) and the other encoding D5-GFP (1.5 micrograms), andincubated for 48 hours.

Approximately 70% of the cells transfected with D1-NLS and D5-GFP hadcell surface expression of D5-GFP, 20% of the cells had both surface andcytoplasm expression of D5-GFP, and 10% had nuclear expression ofD5-GFP. There was no nuclear translocation of the D5 dopamine receptorcoexpressed with D1-NLS, indicating that the D1 and D5 receptors did notoligomerise.

Example 9 D1 Dopamine Receptor Containing Two NLS Motifs (D1-2NLS-RFP)and Treated with Antagonist

By modifying the construct encoding D1-NLS-RFP, a DNA construct(D1-2NLS-RFP) was created to introduce a second NLS into the carboxyltail of the dopamine D1 receptor by replacing the KKEEA sequence of thewild type D1 dopamine receptor with the NLS, KKKRK.

HEK cells were transfected with DNA encoding this construct(D1-2NLS-RFP), and treated at intervals with the antagonist SCH-23390(10 μM) as previously described. At 6, 22, 30 and 42 hours aftertransfection, the culture medium containing antagonist was replaced.Control cells received no antagonist.

In both COS and HEK cells transfected with D1-2NLS-RFP, the receptor waslocated in the nucleus in 100% of cells after 24 hours, indicatingenhanced nuclear translocation when a second NLS was present.

At 48 hours, 90% of cells not treated with antagonist showedfluorescence in the nucleus and 0% of cells had fluorescence on the cellsurface. Antagonist-treated cells showed 51% of cells with cell surfacelabel and 49% with nuclear label.

Incorporation of a second NLS resulted in a more efficient transport ofthe receptor to the nucleus, and this event was still retarded byantagonist treatment.

Example 10 D2 Dopamine Receptor (D2-GFP)

HEK cells were transfected with DNA constructs encoding D2-GFP (2micrograms) and DsRed-NUC (1 microgram), and incubated for 48 hours.Cells were examined by confocal microscopy.

Approximately 90% of the cells expressing D2-GFP had cell surfaceexpression, and 10% had nuclear or cytoplasm expression. The D2 dopaminereceptor, having no endogenous NLS, is predominantly expressed on thecell surface.

Example 11a Dopamine D1 Receptor with an Incorporated NLS (D1-NLS) andDopamine D2 (D2-GFP)

HEK cells were transfected with DNA constructs encoding D1-NLS (7micrograms), and D2-GFP (1.5 micrograms) and incubated for 48 hours. Thecells were also transfected with Ds-Red-NUC to verify the localisationof the nucleus (1 microgram). The cells were examined by confocalmicroscopy.

In cells transfected with D1-NLS and D2-GFP, 33% of the cells had D2-GFPexpression in the nucleus, indicating transport of both D1-NLS andD2-GFP to the nucleus, due to oligomerisation between the D1 and D2receptors. 67% of the cells had D2-GFP receptors on the cell surfaceonly or on surface and cytoplasm.

Example 11b Ability of D2 Dopamine Receptor D2 Short (D2S) to Dimerisewith Dopamine Receptor D2 Long (D2L)

HEK cells were transfected with DNA constructs encoding D2S-GFP (2micrograms) and D2L-NLS (2 micrograms) and were incubated for 48 hours.Nuclei were visualised with DsRED-NUC (2 micrograms). The cells wereexamined by confocal microscopy.

D2S-GFP receptor was visualised in the nuclei of 29% of cells. Thisindicated that D2S dimerised with D2L and was transported to thenucleus.

Example 11c Ability of Dopamine Receptor D2S to Dimerise with DopamineReceptor D2L

HEK cells were transfected with DNA constructs encoding D2S-RFP (2micrograms) and D2L-NLS-GFP (2 micrograms) and incubated for 48 hours.The cells were examined by confocal microscopy.

40% of the cells had a yellow colour (red plus green overlay) in thenucleus, indicating that D2L-NLS dimerised with D2S-RFP and transportedit to the nucleus.

Example 12 D2 Dopamine Receptor with an Incorporated NLS (D2-NLS-GFP)Treated with Antagonists

HEK cells were transfected with DNA encoding D2-NLS-GFP and the cellswere treated with the D2 dopamine receptor antagonists, (+)butaclamol(10 micromolar) or raclopride (10 micromolar). At 6, 22, 30 and 42 hoursafter transfection, the cells were treated with the antagonists. Cellswere incubated 48 hrs after drug treatment and examined by confocalmicroscopy.

In the absence of antagonist, cells expressing D2-NLS-GFP showed nuclearlabel in 70% of cells, and cytoplasmic labelling in 20% and cytoplasmicand surface labelling in 10% of cells. With (+)butaclamol treatment,nuclear labelling appeared in only 5% of cells, 5% of cells hadcytoplasmic label and 90% of the cells had cell surface labelling. Withraclopride treatment, 5% of cells showed nuclear labelling, 15% of cellshad cytoplasmic labelling and 80% of cells had cell surface labelling.Both antagonists of the D2 receptor prevented the translocation of thereceptor off the cell surface and to the nucleus.

Example 13 Beta2-adrenergic Receptor-GFP (beta2-AR-GFP)

A DNA construct was created encoding a fusion protein comprising thehuman beta2-adrenergic receptor and GFP (beta2-AR-GFP). Cells weretransfected with the DNA construct encoding beta2-AR-GFP (2 micrograms),and incubated for 24 hours and examined by confocal microscopy.

In cells expressing beta2-AR-GFP, 42% of cells had receptor expressionin cytoplasm only, and 58% of the cells had receptor expression in thecytoplasm and on the cell surface. No nuclear localisation of thereceptor was observed.

Example 14 Beta2-adrenergic Receptor with an Incorporated NLS(beta2-AR-NLS3-GFP)

A DNA construct was created encoding a fusion protein comprising thehuman beta2-AR-NLS3-GFP. HEK cells were transfected with DNA encodingbeta2-AR-NLS3-GFP-3 (2 microgram), and Ds-Red-NUC (1 microgram) and thecells were incubated for 48 hours.

45% of the cells transfected with beta2-AR-NLS3-GFP had nuclearlocalisation of receptor and 55% of the cells had surface andcytoplasmic expression. Incorporation of a NLS into the beta2-AR inducedreceptor translocation to the nucleus.

Example 15 Beta2-adrenergic Receptor with an Incorporated NLS(beta2-AR-NLS3-GFP), Treated with Antagonist

HEK cells were transfected with DNA encoding beta2-AR-NLS3-GFP (1microgram), and Ds-Red-NUC (1 microgram) and incubated for 48 hours.Cells were treated at intervals with atenolol (10 micromolar), anadrenergic receptor antagonist. At 6, 22, 30 and 42 hours aftertransfection, the culture medium containing the antagonist was replaced.

Control cells received no antagonist. In control cells, 60% had receptorexpression in the nucleus, 21% had receptor expression on the cellsurface, 19% had receptor expression in the cytoplasm.

In antagonist atenolol-treated cells, 70% had receptor expression on thecell surface, 14% had receptor expression in the nucleus, and 16% hadreceptor expression in the cytoplasm. Treatment with the antagonistatenolol prevented beta2-AR-NLS3-GFP trafficking to the nucleus andretained the receptor on the cell surface.

Example 16 Beta2-adrenergic Receptor (beta2-AR-GFP) Coexpressed withDopamine D1 Receptor with an Incorporated NLS (D1-NLS)

HEK cells were transfected with DNA constructs encoding the beta2-AR-GFP(1.5 microgram) and D1-NLS (3 microgram) for 48 hours.

Approximately 40% of cells showed beta2-AR-GFP receptor expression inthe nucleus, demonstrating that the beta2-AR not containing an NLS hadtrafficked to the nucleus. This indicated that oligomerisation hadoccurred between the beta2-AR receptor and D1 dopamine receptor,containing the NLS. 45% of the cells showed beta2-AR-GFP in thecytoplasm and 15% in cytoplasm and on cell surface.

Example 17 Beta2-Adrenergic Receptor (beta2-AR-GFP) and Dopamine D1Receptor with an Incorporated NLS (D1-NLS) Treated with Antagonist

HEK cells were transfected with DNA constructs encoding beta2-AR-GFP(1.5 micrograms) and D1-NLS (3 micrograms) and incubated for 48 hours.These cells were treated with the adrenergic antagonist, propranolol (5micromolar). At 6, 22, 30 and 42 hours after transfection the culturemedium containing the antagonist was replaced. Control cells received noantagonist. The cells were examined by confocal microscopy.

25% of control cells showed beta2-AR-GFP nuclear expression and 75% ofcells showed label in cytoplasm and on cell surface.

In propranolol-treated cells, beta2-AR-GFP nuclear expression was 10%,and 90% of the cells showed cytoplasmic and surface label. The formationof a heterooligomer with between beta2-AR-GFP and D1-NLS resulted in thetrafficking of the beta2-AR-GFP to the nucleus. This trafficking wasattenuated by the presence of the antagonist to the adrenergic receptor.

Example 18 Beta2-adrenergic Receptor with an Incorporated NLS(beta2-AR-NLS3-GFP)

HEK cells were transfected with a DNA construct containing an NLSencoding beta2-AR-NLS3-GFP (8 micrograms) for 48 hours. The cells werealso transfected Ds-Red-NUC to verify the localisation of the nucleus (1microgram).

80% of cells showed beta2-AR-NLS3-GFP receptor in the nucleus. Theefficiency of the NLS was improved, resulting in a greater localisationof receptor in the nucleus.

Example 19 Serotonin 1B Receptor with an Incorporated NLS(5HT1B-NLS-GFP) and Treatment with Antagonist

HEK cells were transfected with a DNA construct encoding the serotonin5HT1B-NLS-GFP (2 micrograms). The cells were transfected with Ds-red-NUCto verify the localisation of the nucleus (1 microgram). Cells weretreated with methysergide (10 micromolar), a serotonin receptorantagonist. At 6, 22, 30 and 42 hours after transfection, the culturemedium containing antagonist was replaced. Control cells received noantagonist. The cells were examined by confocal microscopy.

Control cells, not treated with antagonist, showed 55% with receptorlocalised in the nucleus, and 20% with receptor localised on the cellsurface. At 48 hours, methysergide-treated cells showed 25% of the cellshad receptor in the nucleus and 62% of the cells had cell surfacelocalisation.

The serotonin 5HT1B receptor was efficiently translocated from the cellsurface to the nucleus by insertion of the NLS. Treatment with theserotonin antagonist methysergide prevented the translocation of thereceptor.

Example 20 Cysteinyl Leukotriene Receptor-2 with an Incorporated NLS(CysLT2-NLS-GFP)

HEK cells were transfected for 48 hours with a DNA construct encodingCysLT2-NLS-GFP (8 micrograms). The cells were also transfected withDs-RED-NUC to verify the localisation of the nucleus (1 microgram). Thecells were examined by confocal microscopy.

83% of cells expressing Cys-LT2-NLS-GFP showed receptor expression inthe nucleus and 0% of cells had receptor expression on the cell surface,indicating Cys-LT2-NLS-GFP receptor localisation in the nucleus.

Example 21 Cysteinyl Leukotriene Receptor-2 with an Incorporated NLS(Cys-LT2-NLS-GFP) Treated with Antagonist

The DNA encoding Cys-LT2-NLS-GFP (3 micrograms) was used to transfectHEK cells. These cells were treated with the cysteinyl leukotrienereceptor antagonist, montelukast (10 micromolar). At 6, 22, 30 and 42hours after transfection the culture medium containing antagonist wasreplaced. Control cells received no antagonist. The cells were examinedby confocal microscopy.

In the absence of antagonist, 70% of cells expressing Cys-LT2-NLS-GFPhad localisation of receptor in the nucleus and 30% of cells showed acytoplasmic localisation with 0% of cells showing receptor on the cellsurface. For the antagonist-treated cells, only 10% showed nuclearlocalisation of the receptor, while 90% showed cell surface expressionof receptor. Thus the cysteinyl leukotriene receptor antagonistmontelukast prevented the transport of the Cys-LT2-NLS-GFP receptor offthe cell surface and into the nucleus.

Example 22 Mu opioid Receptor with an Incorporated NLS (muopioid-NLS-GFP)

HEK cells were transfected for 48 hours with a DNA construct encodingthe mu opioid-NLS-GFP (2 micrograms). The cells were also transfectedwith Ds-Red-NUC (1 microgram) to verify the localisation of the nucleus.The cells were examined by confocal microscopy.

65% of the mu opioid-NLS-GFP transfected cells showed receptorexpression in the nucleus. 15% of the cells showed cell surfacelocalisation of receptor and 20% receptor of cells had cytoplasmiclabelling. Thus the insertion of the NLS permitted the mu opioidreceptor to traffic to the nucleus.

Example 23 Mu opioid Receptor with an Incorporated NLS (mu-NLS-GFP)Treated with Antagonists

HEK cells were transfected with a DNA construct encoding the muopioid-NLS-GFP (2 micrograms). The transfected cells were treated withthe mu opioid antagonists, naloxone (10 micromolar) or naltrexone (10micromolar). At 6, 22, 30 and 42 hours after transfection the culturemedium containing the antagonist was replaced. Control cells received noantagonist. The cells were examined by confocal microscopy.

When untreated, 62% of cells had Mu-NLS-GFP in the nucleus and 20% ofcells had receptor detectable on the cell surface. With naloxonetreatment, 21% of cells had receptor expression in the nucleus and 66%of cells had receptor on the cell surface. With naltrexone treatment,22% of cells had receptor expression in the nucleus and 58% of cells hadreceptor on the cell surface. Thus the mu opioid antagonists naloxoneand naltrexone reduced receptor translocation off the cell surface andto the nucleus.

Example 24 Muscarinic M1 Receptor with an Incorporated NLS (M1-NLS-GFP)Treated with Antagonist

HEK cells were transfected with DNA encoding M1-NLS-GFP (1 microgram),and Ds-Red-NUC (1 microgram) for 48 hours. These cells were treated withiprotropium bromide (10 micromolar). The medium containing antagonistwas replaced at 6, 22, 30 and 42 hours after transfection. Control cellsreceived no antagonist treatment. The cells were examined by confocalmicroscopy.

Following iprotropium bromide treatment, 72% of the cells had receptorexpression on the cell surface, 17% had receptor expression in thecytoplasm only, 11% of the cells had receptor expression in the nucleus.

For control cells, 64% had receptor expression in the nucleus, 23% hadreceptor expression on the cell surface and 13% of the cells hadreceptor expression in cytoplasm.

Treatment with a muscarinic antagonist prevented the M1-NLS-GFP fromtranslocating off the cell surface and trafficking to the nucleus.

Example 25 Histamine H1 Receptor with an Incorporated NLS (H1-NLS-GFP)

HEK cells were transfected with a DNA construct encoding the histamineH1-NLS-GFP receptor (2 micrograms), and a construct encoding Ds-Red-NUC(1 microgram) for and incubated for 48 hours.

Approximately 65% of the cells had receptor expression in the nucleus,and 35% of the cells had receptor expression on both surface andcytoplasm. Insertion of the NLS into the H1 histamine receptor resultedin translocation of the receptor off the surface and to the nucleus.

Example 26 Effect of the Antagonist Promethazine on the Trafficking ofthe H1 Histamine Receptor with an NLS Inserted (H1-NLS-GFP)

HEK cells were transfected with H1-NLS-GFP (2 micrograms) and DsRED-Nuc(2 micrograms) and incubated for 48 hours. The cells were treated withpromethazine (10 micromolar) for 48 hours. Nucleii were visualised withDsRED-Nuc. The cells were examined by confocal microscopy.

With the promethazine treated cells 88% of the cells had receptor on thecell surface, 10% of the cells had receptor in the nucleus. With theuntreated cells 85% had receptor expression in the nucleus andcytoplasm. Thus treatment with promethazine reduced H1-NLS-GFPtrafficking to the nucleus.

Example 26 Angiotensin AT1 Receptor (AT1R)

A DNA construct (AT1R-RFP) was created encoding a fusion proteincomprising the NLS-containing human angiotensin AT1 receptor and DsRed2(RFP).

COS cells were transfected with the DNA construct AT1R-RFP (4micrograms) and incubated for 48 hours at 37° C.

Cells were examined by confocal microscopy and the receptor was found tobe located exclusively within the nuclei of the cells, indicating abasal agonist-independent translocation of the AT1R into the nucleus.

Example 27 Dopamine Receptor (D1-NLS-GFP) Treated with Agonist for aShort Term

HEK cells were transfected with the DNA constructs encoding D1-NLS-GFP(2 micrograms), and D1-WT (4 micrograms), and the cells incubated for 24hours. The cells were treated with the dopamine D1 agonist, SKF 81297(10 micromolar) for 35 mins. A single group of cells were visualised byconfocal microscopy in real time.

An increased expression of the receptor in the nucleus was demonstrated,with a maximum increase occurring at 20 minutes, indicating short termagonist effect.

Example 28 Dopamine Transporter with a NLS, Fused to GFP and RFP(DAT-NLS-GFP and DAT-NLS-RFP)

HEK cells were transfected with a DNA construct encoding DAT-GFP (2micrograms) for 48 hours. Nuclei were visualised with DsRED-nuc (2micrograms) using confocal microscopy.

At 48 hours, DAT-GFP was detected on the cell surface or in thecytoplasm in 86% of the cells. In 14% of the cells, the transporter wasin the nucleus.

HEK cells were transfected with a construct encoding DAT-NLS-RFP (2micrograms) and visualised by confocal microscopy at 48 hours.DAT-NLS-RFP was detected in the nuclei in 85% of the cells. In 18% ofthe cells, the transporter was either at the surface or in thecytoplasm.

HEK cells were then transfected with DNA encoding DAT-NLS-GFP. (2micrograms) and visualised by confocal microscopy at 48 hours. Nucleiwere visualised with DsRED-nuc (2 micrograms). DAT-NLS-GFP was detectedin the nucleus of 77% of cells.

Example 29 Co-Trafficking of DAT-GFP with DAT-NLS-RFP

HEK cells were transfected with DNA constructs encoding DAT-NLS-RFP (2micrograms) and DAT-GFP (2 micrograms), and incubated for 48 hours. Thecells were examined by confocal microscopy.

A yellow fluorescence was detected in the nuclei in 56% of the cells,indicating co-localisation of DAT-NLS-RFP and DAT-GFP in the nucleus,confirming oligomerisation of DAT-NLS-RFP and DAT-GFP.

Example 30 Effect of Cocaine on DAT-NLS-RFP Trafficking to the Nucleus

HEK cells were transfected with a DNA construct encoding DAT-NLS-RFP (2micrograms) and incubated for 48 hours. At 6, 22, 30 and 42 hours aftertransfection, the cells were treated with cocaine or amphetamine (finalconcentration 10 micromolar), or left untreated. The cells were examinedby confocal microscopy.

In the non-treated HEK cells, 77% of the cells had DAT-NLS-RFPexpression in the nucleus.

Following cocaine treatment, 75% of the cells had cell surface orcytoplasmic expression of DAT-NLS-RFP, whereas 25% of the cells hadtransporter expression in the nucleus and cytoplasm. Treatment withcocaine reduced the trafficking of the DAT-NLS-RFP to the nucleus.

Following amphetamine treatment, 34% of the cells had cellsurface/cytoplasm expression, and 66% of the cells had transporterexpression in the nucleus/cytoplasm. Treatment with an amphetamine(which does not target DAT but targets the vesicular monoaminetransporter, VMAT) had no inhibitory effect on the trafficking of theDAT-NLS-RFP to the nucleus.

Example 31 Expression of the Dopamine Transporter with a NLS(DAT-NLS-GFP) and Treatment with Antagonists

HEK cells were transfected with a DNA construct encoding DAT-NLS-GFP (2micrograms) and incubated for 48 hours. At 6, 22, 30 and 42 hours aftertransfection, the cells were treated with GBR-12909 (final concentration1 micromolar). The cells were examined by confocal microscopy.

HEK cells were transfected with a construct encoding DAT-NLS-GFP (2micrograms) and incubated for 48 hours. At 6, 22, 30 and 42 hours aftertransfection cells were treated with mazindol (final concentration 1micromolar). The cells were examined by confocal microscopy.

Control HEK cells were transfected with DAT-NLS-GFP incubated for 48hours and not treated with drug.

In the untreated HEK cells transfected with DAT-NLS-GFP, 77% of thecells had transporter expression in the nucleus and cytoplasm, 23% inthe cytoplasm only, and 0% on the cell surface.

Following GBR-12909 treatment, 62% of the cells had transporterexpression on the cell surface and in cytoplasm, and 38% of the cellshad transporter expression in the nucleus and cytoplasm. Treatment withGBR-12909 reduced DAT-NLS-GFP translocation off the cell surface andtrafficking to the nucleus.

Following mazindol treatment 61% of the cells had cell surface andcytoplasm expression of transporter, and 39% of the cells hadtransporter expression in the nucleus and cytoplasm. Treatment withmazindol reduced the DAT-NLS-GFP translocation of the cell surface andtrafficking to the nucleus.

Example 32 Ability of Dopamine Transporter to Homooligomerise, UsingStaggered Expression of DAT-GFP and DAT-NLS-RFP

HEK cells were transfected with the DNA construct encoding DAT-GFP (2micrograms) and 24 hrs later with the DNA construct encoding DAT-NLS-RFP(0.5, 1, and 2 micrograms) and incubated for 48 hours. Cells were alsotransfected with DAT-GFP alone as control. The cells were incubated 48hours after the second transfection. Total incubation period was 72hours.

85% of the cells transfected with DAT-GFP alone contained transporter inthe cytoplasm, and 7% in the nucleus. In the staggered experiment (ratio1:0.5), 97% of the cells had yellow (=red+green) fluorescence in thenucleus. In the staggered experiment (ratio 1:1), 94% of the cells hadyellow fluorescence in the nucleus. In the staggered experiment (ratio1:2), 94% of the cells had yellow fluorescence in the nucleus. Thereforethe DAT-GFP interacted with and dimerised with DAT-NLS-RFP in order totraffic to the nucleus.

Example 33 Expression of the Metabotropic Glutamate-4-Receptor(mGluR4-GFP)

HEK cells were transfected with a DNA construct encoding mGluR4-GFP (2micrograms) and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

89% of the receptors were expressed at the cell surface. Thus the mGluR4receptor was largely located at the cell surface.

Example 34 Expression of the Metabotropic Glutamate-4 Receptor with anInserted NLS (mGluR4-NLS-GFP)

HEK cells were transfected with a DNA construct encoding mGluR4-NLS-GFP(2 micrograms), and incubated for 48 hours. The cells were alsotransfected with Ds-RED-NUC (2 micrograms) to verify the localisation ofthe nucleus. The cells were examined by confocal microscopy.

60% of cells expressing mGluR4-NLS-GFP showed expression of receptor inthe nucleus.

Thus the insertion of an NLS into the mGluR4 receptor increased thenuclear localisation of the receptor.

Example 35 Expression of the Muscarinic M1 Receptor with or without NLSIncorporation (M1-GFP and M1-NLS-GFP)

HEK cells were transfected with a DNA construct encoding the M1-GFP (2micrograms) or with a construct encoding the M1-NLS-GFP (2 micrograms)and incubated for 48 hours. Nuclei were visualised with DsRED-NUC (2micrograms). The cells were examined by confocal microscopy.

After transfection with M1-GFP, 67% of the cells had receptor expressedon the cell surface or in the cytoplasm.

Transfection with M1-NLS-GFP showed 92% of the cells with nuclearexpression of the receptor, indicating that the NLS directed thereceptor to the nucleus.

Example 36 Expression of the H1 Histamine Receptor (H1-GFP)

HEK cells were transfected with a DNA construct encoding H1-GFP (1.5micrograms) and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

97% of the cells expressed receptor at the cell surface. Thus, theunmodified receptor did not traffic to the nucleus.

Example 37 Expression of the Cysteinyl Leukotriene Receptor with NLSInserted (CysLT1-NLS-GFP)

HEK cells were transfected with a DNA construct encoding CysLT1-NLS-GFP(2 micrograms) and incubated for 48 hours. Nuclei were visualised withDsRED-NUC (2 micrograms). The cells were examined by confocalmicroscopy.

With the control untreated cells, 0% of the cells had receptorexpression on the cell surface, and 100% of the cells had nuclearexpression, indicating robust removal of the receptor off the cellsurface.

Example 38 Expression of the Serotonin Transporter Fused to GFP(SERT-GFP)

HEK cells were transfected with a DNA construct encoding SERT-GFP (2micrograms) and incubated for 48 hours. 91% of the cells expressedtransporter on the cell surface and cytoplasm.

Example 39 Expression of the Serotonin Transporter with an Inserted NLSand Treatment with Fluoxetine (SERT-NLS-GFP)

HEK cells were transfected with DNA encoding SERT-NLS-GFP (2 microgramsof DNA) and treated with fluoxetine (final concentrations micromolar)for 48 hours. At 6, 22, 30 and 42 hours after transfection, the cellswere treated with fluoxetine (final concentration 1 micromolar). Thecells were examined by confocal microscopy.

In the untreated cells expressing SERT-NLS-GFP, 0% of the cells hadtransporter expression on the cell surface, 26% had transporterexpression in the cytoplasm, and 60% of the cells had transporterexpression in the nucleus and cytoplasm.

Following fluoxetine treatment, 68% of the cells had SERT-NLS-GFPtransporter expression on the cell surface and cytoplasm, and 27% of thecells had transporter expression in the nucleus and cytoplasm. Thustreatment with fluoxetine inhibited the SERT-NLS-GFP from translocatingoff the cell surface and trafficking to the nucleus.

Example 40 Evaluation of the Ability of Two Different Cell SurfaceMembrane Proteins to Interact with Each Other (D2-GFP and DAT-NLS-RFP)

HEK cells were cotransfected with DNA constructs encoding the D2-GFP (2micrograms) and DAT-NLS-RFP (2 micrograms) and incubated for 48 hours.Cells were also transfected separately with D2-GFP and DAT-NLS-RFP aloneas controls. The cells were examined by confocal microscopy.

85% of the cells transfected with DAT-NLS-RFP contained transporter inthe nucleus. 97% of the cells transfected with D2-GFP contained thereceptor on the cell surface, and 4% of the cells contained receptor inthe nucleus. 86% of the cotransfected cells contained yellow (red plusgreen) fluorescence in the nucleus, indicating the presence of both D2and DAT proteins in the nucleus and confirming dimerisation of theco-expressed proteins.

Example 41 Evaluation of the Ability of a Membrane Protein and aNon-Membrane Protein, to Associate in a Complex and Interact with EachOther (D1-NLS and beta-arrestin1-GFP)

HEK cells were co-transfected with DNA constructs encoding D1-NLS (2micrograms) and beta-arrestin1-GFP (2 micrograms) and incubated for 48hours. Cells were also transfected with beta-arrestin1-GFP alone. Thecells were checked by confocal microscopy.

100% of cells transfected with beta-arrestin1-GFP alone expressedfluorescent protein in the cytoplasm. Of these cells 15% also hadfluorescence in the nucleus. 89% of cells co-transfected with bothproteins expressed fluorescent protein in the nucleus and of these 16%had expression in the cytoplasm. Thus, the interaction between the GPCRand the non-membrane protein enabled the trafficking of the non-NLScontaining beta-arrestin protein to the nucleus.

Example 42 Expression of the Dopamine D1 Receptor with an Inserted NLS(HA-D1-NLS) Treatment with Antagonist and Detection with Fluorometry

Wells in a multi-well plate were coated with poly-L-ornithine and thenplated with 50,000 cells per well. The cells were transfected with DNAencoding an HA epitope tagged D1-NLS receptor and treated with(+)butaclamol (10 nanomolar to 10 micromolar) over 48 hours. Followingthis, the cells were fixed with paraformaldehyde, and cell surfacereceptors were detected with a rat anti-HA antibody and then a goatanti-rat antibody conjugated to FITC. The fluorescent signal wasdetected by fluorometry (Cytofluor). The results are the average of fivewells per experimental condition and are shown in FIG. 2. There was adose-dependent effect of butaclamol to retain receptor on the cellsurface, indicating that this antagonist reduced receptor traffickingfrom the cell surface. Thus fluorometry can be utilised to detectreceptor retained at the cell surface.

Example 43 Expression of the Dopamine D1 Receptor with an Inserted NLS(HA-D1-NLS), and Blockade of Antagonist Dose-Response Effect by Agonistand Detection with Fluorometry

Wells in a multi-well plate were coated with poly-L-ornithine and thenplated with 50,000 cells per well. The cells were transfected with DNAencoding an HA epitope tagged D1-NLS receptor and treated with theantagonist SCH 23390 (1 nanomolar to 1 micromolar) with or without theagonist SKF 81297 (1 micromolar) over 48 hours. Following this, thecells were fixed with paraformaldehyde, and cell surface receptors weredetected with a rat anti-HA antibody and then a goat anti-rat antibodyconjugated to FITC. The fluorescent signal was detected by fluorometry(Cytofluor). The results are the average of five wells per experimentalcondition. There was a dose-dependent effect of SCH 23390 to retainreceptor on the cell surface, indicating that this antagonist reducedreceptor trafficking from the cell surface. The concomitant addition ofagonist reduced the antagonist effect (FIG. 3). Thus agonist action canbe detected by blockade of antagonist effect and fluorometry can beutilised to quantify the agonist effect.

Example 43b Expression of the Dopamine D1 Receptor with an Inserted NLS(HA-D1-NLS), and Blockade of Antagonist Effect by Agonist Dose-Responseand Detection with Fluorometry

HEK cells were transfected with HA-D1-NLS in a multi-well plate werecoated with poly-L-ornithine at a concentration of 50,000 cells perwell. The cells were treated with the antagonist SCH 23390 (0.5micromolar) for 48 hrs. The agonist SKF 81297 (100 nanomolar to 1micromolar) together with SCH 23390 was added for the last hour ofincubation. Following this, the cells were fixed with paraformaldehyde,and cell surface receptors were detected with a rat anti-HA antibody andthen a goat anti-rat antibody conjugated to FITC. The fluorescent signalwas detected by fluorometry (Cytofluor). The results are the average offive wells per experimental condition.

Treatment with SCH 23390 retained HA-D1-NLS on the cell surface (FIG. 4,Bar 1 vs. Bar 6). Short-term addition of the agonist resulted in adose-dependent blockade of SCH 23390 effect. Removal of SCH 23390 fromthe cells for the last hour of incubation (and in the absence ofagonist) resulted in a 33% loss of HA-D1-NLS receptors from the cellsurface (FIG. 4, Bar 5 vs. Bar 6), whereas addition of agonist SKF 81297100 nanomolar in the continued presence of SCH 23390 resulted in a 66%loss of receptors from the cell surface (FIG. 4, Bar 4 vs. Bar 6), up toa 78% loss of receptors with addition of SKF 81297 1 micromolar (FIG. 4,Bar 2 vs. Bar 6).

The effect of the antagonist SCH 23390 resulted in retention of receptoron the cell surface, indicating that this antagonist reduced receptortrafficking from the cell surface. The concomitant addition of agonistreduced the antagonist effect and accelerated the removal of thereceptor from the cell surface in a dose-responsive manner. Thusinteracting compounds can be detected by blockade of the effect ofcompounds that retain the NLS-containing receptor at the cell surfaceand fluorometry can be utilized to quantify the effect.

Example 44 Expression of the Dopamine D1 Receptor with an Inserted(D1-NLS), Treatment with (+)butaclamol 10 Micromolar and Detection withRadioligand Binding

HEK cells were transfected with DNA encoding D1-NLS and treated with(+)butaclamol (10 micromolar) or left untreated. After 48 hours, thecells were washed, harvested, lysed and homogenised by polytron. Themembrane fraction was collected by centrifugation and then layered over35% sucrose solution and centrifuged, at 30,000 rpm at 4 degrees C. for90 min to collect the heavy membrane fraction.

The membrane fractions were subjected to radioligand binding assay using[³H]-SCH23390 with (+)butaclamol (10 micromolar) used to define specificbinding. The incubation was at room temperature for 2 hours, followed byrapid filtration and quantitation by scintillation counting.

Antagonist treatment of D1-NLS prevented its translocation off the cellsurface and to the nucleus and the receptor retained on the cell surfacewas quantified by radioligand binding assay (FIG. 5).

Example 45 Expression of the Dopamine D1 Receptor with an Inserted NLS(D1-NLS), Treatment with (+)butaclamol 500 Nanomolar and Detection withRadioligand Binding

HEK cells were transfected with DNA encoding D1-NLS and treated with(+)butaclamol 500 nanomolar or left untreated. After 48 hours, the cellswere washed, harvested, lysed and homogenised by polytron. The membranefraction was collected by centrifugation and then layered over 35%sucrose solution and centrifuged at 30,000 rpm at 4 degrees C. for 90min to collect the heavy membrane fraction.

The membranes were subjected to radioligand binding assay using[³H]-SCH23390 with (+)butaclamol 10 micromolar used to define specificbinding. The incubation was at room temperature for 2 hours, followed byrapid filtration and quantitation by scintillation counting. Results areshown in Tables 4 and 5.

TABLE 4 Control plasma membrane fraction Sample 1 Sample 2 Mean NSB SBpmol/mg prot 2739 3596 3167 1077 2090 1.42

TABLE 5 Butaclamol treated plasma membrane fraction Sample 1 Sample 2Mean NSB SB pmol/mg prot 16419 15362 15890 471 15419 13.15 NSB:non-specific binding SB: specific bindingAntagonist treatment with (+)butaclamol of D1-NLS prevented itstranslocation to the nucleus and the receptor retained on the cellsurface was quantified by radioligand binding assay.

Example 46 Expression of the Dopamine D1 Receptor with an Inserted NLS(D1-NLS), Treatment with (+)butaclamol 100 Nanomolar and Detection withRadioligand Binding

HEK cells were transfected with DNA encoding D1-NLS and treated with(+)butaclamol 100 nanomolar or left untreated. After 48 hours, the cellswere washed, harvested, lysed and homogenised by polytron. The membranefraction was collected by centrifugation and then layered over 35%sucrose solution and centrifuged at 30,000 rpm at 4 degrees C. for 90min to collect the heavy membrane fraction.

The membranes were subjected to radioligand binding assay using[³H]-SCH23390 with (+)butaclamol (10 micromolar) to define specificbinding. The incubation was at room temperature for 2 hours, followed byrapid filtration and quantitation by scintillation counting.

Antagonist treatment with (+)butaclamol (100 nanomolar) prevented D1-NLStranslocation to the nucleus and the receptor retained on the cellsurface was quantified by radioligand binding assay. In the absence ofbutaclamol treatment, 0.03 pmol/mg protein of receptor was detected inthe cell surface membranes, and with butaclamol treatment, 0.09 pmol/mgprotein of receptor was detected in the cell surface membranes.

Example 47 Expression of the Epidermal Growth Factor Receptor (TyrosineKinase Receptor) EGFR-GFP and EGFR-NLS-GFP

HEK cells were transfected with DNA encoding EGFR-NLS-GFP (2micrograms). HEK cells were also transfected with DNA encoding EGFR-GFP(2 micrograms) and incubated for 24 hours.

EGFR-GFP was expressed on the cell surface in 73% of cells and 12% ofcells had receptor in the nucleus. EGFR-NLS-GFP was expressed in thenucleus in 91% of cells and 0% of cells had receptor on the cellsurface. The incorporation of a NLS into the sequence of the EGFreceptor induced robust translocation off the cell surface and into thenucleus.

Example 48 Expression of the Low Density Lipoprotein Receptor (LDL-GFP)

HEK cells were transfected with DNA encoding the LDL-GFP (2 micrograms)and incubated for 24 hours. The receptor was expressed on the cellsurface in 67% of cells and in the nucleus in 8% of cells.

The LDL receptor is expressed on the cell surface in the majority ofcells with not many cells containing receptor in the nucleus.

Example 49 Expression of the LDL Receptor with a NLS (LDL-NLS-GFP)

HEK cells were transfected with DNA encoding LDL-NLS-GFP (2 micrograms),and DsRED-NUC (2 micrograms), and incubated for 48 hours. Cells wereexamined by confocal microscopy.

LDL-NLS-GFP was expressed in the nucleus in 22% of cells, and on thecell surface in 67%. The incorporation of a NLS into the LDL receptorinduced receptor translocation into the nucleus.

Example 50 Expression of the Erythropoietin Receptor (Cytokine Receptor)EPO-GFP and EPO-NLS-GFP

HEK cells were transfected with DNA encoding EPO-NLS-GFP (2 micrograms).HEK cells were transfected with EPO-GPF (2 micrograms). The cells werealso transfected with DsRed-NUC (2 micrograms). The cells were incubatedfor 48 hours and were examined by confocal microscopy.

The EPO-NLS-GFP was located in the nucleus of 72% of cells and on thecell surface in 0% of cells. The EPO-GFP was located on the cell surfacein 79%) of cells and 28% of cells had receptor expression in thenucleus. The incorporation of a NLS into the sequence of the EPOreceptor induced translocation off the cell surface and into thenucleus.

Example 51 Expression of the Serotonin Transporter with a NLS(SERT-NLS-GFP) and Treatment with Sertraline

HEK cells were transfected with DNA encoding SERT-NLS-GFP (2 microgramsof DNA) and treated with sertraline (final concentration 500 nanomolar)for 48 hours. At 6, 22, 30 and 42 hours after transfection, the cellswere treated with sertraline. The cells were examined by confocalmicroscopy.

In the untreated cells expressing SERT-NLS-GFP, 0% of the cells hadtransporter expression on the cell surface and 75% of the cells hadtransporter expression in the nucleus and cytoplasm.

Following sertraline treatment, 69% of the cells had SERT-NLS-GFPtransporter expression on the cell surface and cytoplasm, and 21% of thecells had transporter expression in the nucleus and cytoplasm. Thustreatment with sertraline inhibited the SERT-NLS-GFP from translocatingoff the cell surface and trafficking to the nucleus.

Example 52 Expression of D1 Dopamine Receptor with an Alternate NLS(D1-NLS2-GFP) and Treatment with Antagonists

HEK cells were transfected with DNA encoding D1-NLS2-GFP (2 micrograms)and treated with (+)butaclamol or SCH 23390 (1 micromolar) for 48 hrs.Nuclei were visualised with DsRed-nuc (2 micrograms). Cells wereexamined by confocal microscopy.

With butaclamol treatment, 81% of cells had receptor on the cell surfaceor in cytoplasm and 19% of cells had receptor expression in the nucleus.With SCH 23390 treatment, 78% of cells had receptor on the cell surfaceor in cytoplasm and 22% of cells had receptor expression in the nucleus

In the untreated cells, 89% of cells had receptor expression in thenucleus and cytoplasm.

Thus treatment with the dopamine D1 antagonists prevented D1-NLS2-GFPreceptor translocation off the surface and trafficking to or toward thenucleus.

The present invention is not limited to the features of the embodimentsdescribed herein, but includes all variations and modifications withinthe scope of the claims.

REFERENCES

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TABLE 1 EXAMPLES OF NUCLEAR LOCALISATION SEQUENCES (adapted from Jans etal. 2002) (SEQ ID Protein Nuclear Localisation Sequence NO) Human a1T-ag PKKKRKV 129 CBP80 RRR-(11 aa)-KRRK 130 DNA helicase Q1 KK-(15aa)-KKRK 131 BRCA1 KRKRRP, PKKNRLRRK 132, 133 Mitosin KRQR-(20 aa)-KKSKK134 Myc PAAKRVKLD 135 NF-kB p50 QRKRQK 136 NF-kB p65 HRIEEKRKRTYETFKSI137 HIV1422 KKKYKLK 138 HIV1423 KSKKKAQ 139 Human a2 T-ag PKKKRKV 129NF-kB p50 QRKRQK 136 DNA helicase Q1 KK-(15 aa)-KKRK 131 LEF-1 KKKKRKREK140 EBNA1 LKRPRSPSS 141 HIV-1 IN KRK-(22 aa)-KELQKQITK 142 HIV-1 MAGKKKYKLKH 143 HIV1422 KKKYKLK 144 HIV1423 KSKKKAQ 145 RCP 4.1R EED-(350aa)-KKKRERLD 146 Human a3 T-ag PKKKRKV 129 DNA helicase Q1CYFQKKAANMLQQSGSKNTGAKKRK 147 tTS DILRR-(323 aa)-PKQKRK 148 Human a4T-ag PKKKRKV 129 Mouse a1 LEF-1 KKKKRKREK 140 Mouse a2SSDDEATADSQHSTPPKKKRKV 149 T-agáCK2 site Impa-P1) T-ag PKKKRKV 129 N1N2RKKRK-(9 aa)-KAKKSK 150 RB KR-(11 aa)-KKLR 151 Dorsal áPK{hacek over( )}A site RRPS-(22 aa)-RRKRQK 152 CBP80 RRR-(11 aa)-KRRK 153 DNAhelicase Q1 KK-(15 aa)-KKRK 131 LEF-1 KKKKRKREK 140 Mouse a2SSDDEATADSQHSTPPKKKRKV 149 T-agáCK2 Impa-P1) T-ag PKKKRKV 129 N1N2RKKRK-(9 aa)-KAKKSK 150 RB KR-(11 aa)-KKLR 151 Dorsal áPK{hacek over( )}A RRPS-(22 aa)-RRKRQK 152 CBP80 RRR-(11 aa)-KRRK 153 DNA helicase Q1KK-(15 aa)-KKRK 131 LEF-1 KKKKRKREK 140 Xenopus a1 T-ag PKKKRKV 129Nucleoplasmin KR-(10 aa)-KKKL 154 Yeast a1 T-ag PKKKRKV 129 (SRP1,Kap60) SSDDEATADSQHSTPPKKKRKV 149 T-agáCK2 N1N2 RKKRK-(9 aa)-KAKKSK 150HIV-1 IN KRK-(22 aa)-KELQKQITK 142 Plant a1 T-ag PKKKRKV 129 T-agáCK2SSDDEATADSQHSTPPKKKRKV 149 Opaque-2 RKRK-(7 aa)-RRSRYRK 155 R Protein(Maize) MISEALRKA 156 N1N2 RKKRK-(9 aa)-KAKKSK 150 RAG-1, recombinationactivating protein 1; RCP, red cell protein; RB, Retinoblastoma protein;STAT, signal transducer and activator of transcription (TF); CBP80,Cap-binding protein; LEF, Lymphocyte enhancer factor; EBNA, Epstein-Barrvirus nuclear antigen; IN, HIV-1 integrase; tTG, tissuetransglutaminase; ICP, Infected cell protein.

1. A method for screening a candidate compound for its ability tointeract with at least one transmembrane protein comprising:transfecting a eukaryotic cell with at least one nucleotide sequenceencoding a protein comprising a transmembrane protein containing atleast one nuclear localisation sequence (NLS) and a detectable moietyand permitting expression of the encoded protein in the cell; contactingthe cell with a candidate compound; and determining the distribution ofthe expressed protein in the cell by detecting the distribution of thedetectable moiety in the cell; wherein detection of an altereddistribution of the detectable moiety in the cell relative to thedistribution of the detectable moiety in a control cell not contactedwith the candidate compound indicates that the compound interacts withthe transmembrane protein; and wherein the wild type transmembraneprotein lacks an NLS and the nucleotide sequence encoding thetransmembrane protein is modified to encode an NLS.
 2. The method ofclaim 1 wherein the detectable moiety is a detectable peptide comprisingan antigenic portion of the amino acid sequence of the transmembraneprotein and/or wherein the nucleotide sequence encodes a fusion proteincomprising a transmembrane protein containing at least one NLS and adetectable moiety.
 3. The method of claim 1 wherein the nucleotidesequence is modified to encode an NLS selected from Table 1 or whereinthe nucleotide sequence is modified to encode an amino acid sequenceselected from the group consisting of KKFKR (SEQ ID NO: 158), PKKKRKV(SEQ ID NO: 154) and AFSAKKFKR (SEQ ID NO: 159).
 4. The method of claim1 wherein the eukaryotic cell is a selected from the group consisting ofa mammalian cell, selected from the group consisting of HEK, COS and CHOcells, a yeast cell, an insect cell, a nematode cell, a plant cell and afungal cell.
 5. The method of claim 2 wherein the detectable moiety isan antigenic peptide and the distribution of the antigenic peptide inthe cell is determined by allowing it to bind to an antibody-baseddetection system comprising an antibody specific for the antigenicpeptide selected from the group consisting of an antibody-baseddetection system comprising a first antibody specific for the antigenicpeptide and a second antibody carrying a detectable label and specificfor the first antibody and an antibody-based detection system comprisinga first antibody specific for the antigenic peptide and carrying adetectable label, wherein the detectable label is selected from thegroup consisting of an optically detectable label, a luminescent and afluorescent label.
 6. The method of claim 2 wherein the detectablemoiety is a polypeptide selected from the group consisting of greenfluorescent protein, red fluorescent protein and modified variantsthereof.
 7. The method of claim 1 wherein the transmembrane protein isselected from the group consisting of a G protein coupled receptor(GPCR), a transporter, a cytokine receptor, a tyrosine kinase receptorand a low density lipoprotein (LDL) receptor.
 8. The method of claim 7wherein the transmembrane protein is a GPCR.
 9. The method of claim 8wherein the GPCR is selected from the group consisting of a dopamine D1receptor, a dopamine D2 receptor, a dopamine D3 receptor, a dopamine D5receptor, a histamine 1 receptor, a cysteinyl leukotriene receptor 1, acysteinyl leukotriene receptor 2, an opioid receptor, a muscarinicreceptor, a serotonin receptor, a beta2-adrenergic receptor and ametabotropic glutamate 4 receptor.
 10. The method of claim 7 wherein thetransmembrane protein is a transporter selected from the groupconsisting of a dopamine transporter and a serotonin transporter, acytokine receptor selected from the group consisting of anerythropoietin receptor and an insulin receptor, a tyrosine kinasereceptor selected from the group consisting of an epidermal growthfactor receptor and an insulin receptor, and a low density lipoproteinreceptor.
 11. The method of claim 1 wherein the cell is transfected witha plurality of nucleotide sequences, each of said sequences encoding aprotein comprising a different transmembrane protein containing at leastone NLS and wherein each of said nucleotide sequences encodes a proteincomprising a different detectable moiety or wherein at least onedetectable moiety is common to at least two encoded proteins.
 12. Themethod of claim 1 wherein the cell is contacted with a compound known tointeract with the at least one transmembrane protein prior to contactingthe cell with the candidate compound and wherein detection of an altereddistribution of the detectable moiety in the cell relative to thedistribution of the detectable moiety in a control cell contacted withthe compound known to interact with the transmembrane protein but notcontacted with the candidate compound indicates that the candidatecompound interacts with the transmembrane protein.
 13. The method ofclaim 1 wherein detection of an altered distribution of the detectablemoiety in the cell comprises detection of a reduced level or anincreased level of the detectable moiety associated with the cellmembrane.
 14. The method of claim 1 wherein detection of an altereddistribution of the detectable moiety in the cell comprises detection ofa reduced level or an increased level of the detectable moiety in thenucleus of the cell.
 15. A method for screening a candidate compound forits ability to interact with at least one transmembrane proteincomprising: transfecting a eukaryotic cell with at least one nucleotidesequence encoding an NLS-containing transmembrane protein and permittingexpression of the encoded protein in the cell; contacting the cell witha candidate compound; and determining the level of NLS-containingtransmembrane protein remaining at the cell membrane by isolating thecell membrane fraction of the cell, contacting the fraction with alabelled ligand of the transmembrane protein and determining the levelof binding of the ligand to the fraction; wherein detection of analtered level of the transmembrane protein at the cell membrane relativeto the level at the cell membrane in a control cell not contacted withthe candidate compound indicates that the compound interacts with thetransmembrane protein, and; wherein the wild type transmembrane proteinlacks an NLS and the nucleotide sequence encoding the transmembraneprotein is modified to encode an NLS.
 16. The method of claim 15 whereinthe labelled ligand is a radio-labelled ligand.
 17. The method of claim15 wherein the nucleotide sequence is modified to encode an NLS selectedfrom Table 1 or wherein the nucleotide sequence is modified to encode anamino acid sequence selected from the group consisting of KKFKR, PKKKRKVand AFSAKKFKR.
 18. The method of claim 15 wherein the eukaryotic cell isselected from the group consisting of a mammalian cell, selected fromthe group consisting of HEK, COS and CHO cells, a yeast cell, an insectcell, a nematode cell, a plant cell and a fungal cell.
 19. The method ofclaim 15 wherein the transmembrane protein is selected from the groupconsisting of a G protein coupled receptor (GPCR), a transporter, acytokine receptor, a tyrosine kinase receptor and a low densitylipoprotein (LDL) receptor.
 20. The method of claim 19 wherein thetransmembrane protein is a GPCR.
 21. The method of claim 20 wherein theGPCR is selected from the group consisting of a dopamine D1 receptor, adopamine D2 receptor, a dopamine D3 receptor, a dopamine D5 receptor, ahistamine 1 receptor, a cysteinyl leukotriene receptor 1, a cysteinylleukotriene receptor 2, an opioid receptor, a muscarinic receptor, aserotonin receptor, a beta2-adrenergic receptor, and a metabotropicglutamate 4 receptor.
 22. The method of claim 19 wherein thetransmembrane protein is a transporter selected from the groupconsisting of a dopamine transporter and a serotonin transporter, acytokine receptor selected from the group consisting of anerythropoietin receptor and an insulin receptor, a tyrosine kinasereceptor selected from the group consisting of an epidermal growthfactor receptor and an insulin receptor, and a low density lipoproteinreceptor.
 23. The method of claim 15 wherein the cell is transfectedwith a plurality of nucleotide sequences, each of said sequencesencoding a protein comprising a different transmembrane proteincontaining at least one NLS and wherein each of said nucleotidesequences encodes a protein comprising a different detectable moiety orwherein at least one detectable moiety is common to at least two encodedproteins.
 24. The method of claim 15 wherein detection of an altereddistribution of the detectable moiety comprises detection of a reducedlevel or an increased level of the detectable moiety associated with thecell membrane.